Compositions and methods for detection of candida species

ABSTRACT

The invention features compositions and methods for the detection of Candida species in biological samples, such as human tissue samples. The invention features species specific nucleic acid probes that can hybridize to target nucleic acid molecules in Candida species and can be used in a variety of detection assays, such as fluorescence based assays or NMR based assays. The compositions and methods of the invention can be used to rapidly and accurately detect one or more Candida species in patients and can be used to assist in point-of care-decisions for treating Candida infections.

BACKGROUND OF THE INVENTION

Candida species are commensal organisms of humans, usually found on themucous membranes of the gut, oral cavity, and vaginal introitus, and inwarm moist skin folds. Candida is the most commonly identified causativeagent of oral or vaginal thrush. Candida can also cause life-threateninginfections in hospital patients. In particular, Candida can causeinvasive diseases in hosts with altered immunity, such as in patientswith HIV infection, patients that have received organ or bone marrowtransplants, and in patients experiencing neutropenia after cancerimmunotherapy. Patients on intensive regimens of cancer therapy,patients on prolonged broad spectrum antibiotic therapy, patients usinginvasive devices, and patients on prolonged hospital stays are also athigh risk for such infections.

Conventional methods for identifying Candida species in patients includemorphology and assimilation tests involving blood cultures. These testscan be time consuming, laborious, and often produce false negativeresults, which adversely affect the targeting of anti-fungal treatmentand related point-of-care decisions. In addition, these methods mayrequire extensive, specialized equipment and highly trained operators.These methods may further be limited by their sensitivity or the numberof Candida species that can be detected simultaneously.

Accordingly, there is a need for compositions, methods, and kits fordetecting the presence of Candida species in a biological sample in atimely, accurate, and efficient manner.

SUMMARY OF THE INVENTION

The invention features nucleic acid probes and primers that include thesequence of any one of SEQ ID NOs: 1 to 41 (e.g., nucleic acid probeshaving the sequence of any one of SEQ ID NOs: 1 to 10, such as any oneof SEQ ID NOs: 1 to 5), and nucleic acid probes and primers having atleast 90%, 95%, 97%, 99%, or more sequence identity to these probes andprimers.

The nucleic acid probes and primers can be used to detect a Candidaspecies (e.g., Candida albicans, Candida tropicalis, Candida krusei,Candida glabrata, Candida parapsilosis, Candida dubliniensis, Candidalusitaniae, and Candida guillermondi) in a biological sample. In severalembodiments, the nucleic acid probes of the invention include adetection moiety (e.g., a fluorescent label) that is conjugated to theprobe. In other embodiments, the nucleic acid probes are conjugated to amagnetic nanoparticle. In another embodiment, the probes of theinvention (e.g., SEQ ID NOs: 1 to 5) may be molecular beacon probes thatmay include a fluorescent label (e.g., FAM, TAMRA, HEX, TMR, Cy3, Cy5,and other spectrally distinguishable dyes) and a quencher (e.g., DDQ-I,Dabcyl, Eclipse, Iowa Black FQ, BHQ-1, QSY-7, BHQ-2, DDQ-II, Iowa BlackRQ, QSY-21, BHQ-3). In yet another embodiment, the probes of theinvention (e.g., SEQ ID NOs: 1 to 5) may be a “shared-stem” molecularbeacon probe. In other embodiments, the nucleic acid probes of theinvention (e.g., SEQ ID NOs: 6 to 10) may be conjugated to magneticnanoparticles.

The invention features a method for detecting at least one Candidaspecies (e.g., Candida albicans, Candida tropicalis, Candida krusei,Candida glabrata, Candida parapsilosis, Candida dubliniensis, Candidalusitaniae, and Candida guillermondi) in a biological sample (e.g.,soil, water, food, and tissue or fluid sample from an organism, such asa human). The method includes contacting the biological sample with atleast one nucleic acid probe having a sequence selected from any one ofSEQ ID NOs: 1 to 41 (e.g., SEQ ID NOs: 1-10, such as SEQ ID NOs: 1-5)under conditions which allow the at least one probe to hybridize to anucleic acid molecule of the Candida species; and detectinghybridization between the at least one probe and the nucleic acidmolecule, thereby detecting the at least one Candida species. In anembodiment of the method, the probe is a Candida albicans probe and hasthe sequence of SEQ ID NO: 1 or SEQ ID NO: 6; the probe is a Candidatropicalis probe and has the sequence of SEQ ID NO: 2 or SEQ ID NO: 7;the probe is a Candida glabrata probe and has the sequence of SEQ ID NO:3 or SEQ ID NO: 8; the probe is a Candida parapsilosis probe and has thesequence of SEQ ID NO: 4 or SEQ ID NO: 9; the probe is a Candida kruseiprobe and has the sequence of SEQ ID NO: 5 or SEQ ID NO: 10. In severalembodiments, the method features nucleic acid probes that arefluorescently labeled. These nucleic acid probes may be molecular beaconprobes (e.g., probes having the sequence of any one of SEQ ID NOs: 1 to5) that further include a quencher. According to the method of theinvention, hybridization of the nucleic acid probe to target Candidaspecies nucleic acid molecules produces fluorescence which may bedetected in, e.g., a fluorescence based assay (e.g, a real-time orend-point PCR assay) using, e.g., an instrument capable of detectingfluorescence (e.g., a real-time or end-point thermal cycler, or a platereader). In one embodiment, the method features one or more probes(e.g., 2, 3, 4, or 5 or more probes) that may be labeled with the sameor different fluorescent labels (e.g., molecular beacon probes labeledwith spectrally distinguishable fluors), and the one or more probes mayeach be contacted with the sample for use in the same assay, e.g., in amultiplex assay to detect up to five different Candida species (e.g.,Candida albicans, Candida tropicalis, Candida glabrata, Candidaparapsilosis, and Candida krusei) in a biological sample.

The invention also features a method of using nucleic acid probesconjugated to magnetic nanoparticles (e.g., SEQ ID NOs: 6 to 10 and 13to 31) to detect Candida species in, e.g., an NMR based assay (e.g., anaggregation or disaggregation assay) using an NMR instrument. The methodincludes contacting the biological sample with at least one nucleic acidprobe having a sequence selected from any one of SEQ ID NOs: 6 to 31(e.g., SEQ ID NOs: 6-10) under conditions which allow the at least oneprobe to hybridize to a nucleic acid molecule of the Candida species;and detecting hybridization between the at least one probe and thenucleic acid molecule, thereby detecting the at least one Candidaspecies. In an alternate embodiment of the method, probes conjugated tomagnetic nanoparticles can be used in pairwise combinations in an NMRassay to detect Candida species, e.g., a probe having the sequence ofSEQ ID NO: 6 can be used with a probe having the sequence of any one ofSEQ ID NOs: 13 and 14 to detect Candida albicans; a probe having thesequence of SEQ ID NO: 7 can be used with a probe having the sequence ofany one of SEQ ID NOs: 20 to 25 to detect Candida tropicalis; a probehaving the sequence of SEQ ID NO: 8 can be used with a probe having thesequence of any one of SEQ ID NOs: 18 and 19 to detect Candida glabrata;a probe having the sequence of SEQ ID NO: 9 can be used with a probehaving the sequence of any one of SEQ ID NOs: 20 to 25 to detect Candidaparapsilosis; and/or a probe having the sequence of SEQ ID NO: 10 can beused with a probe having the sequence of any one of SEQ ID NOs: 15 to 17to detect Candida krusei. Binding of probes to target Candida nucleicacid molecule can be detected by measuring the NMR relaxation rate in anNMR instrument.

The invention also features primers for DNA sequencing based detectionof Candida species in a biological sample. In several embodiments, theprimer is a Candida krusei primer having the sequence of SEQ ID NO: 32or SEQ ID NO: 33; the primer is a Candida albicans primer having thesequence of SEQ ID NO: 34 or SEQ ID NO: 35; the primer is a Candidaglabrata primer having the sequence of SEQ ID NO: 36 or SEQ ID NO: 37;the primer is a Candida parapsilosis primer having the sequence of SEQID NO: 38 or SEQ ID NO: 39; or the primer is a Candida tropicalis primerhaving the sequence of SEQ ID NO: 40 or SEQ ID NO: 41. Sequencing mayalso be preceded by prior amplification using SEQ ID NO: 11 and SEQ IDNO: 12. Alternatively, the sequencing could be done using SEQ ID NO: 11and SEQ ID NO: 12 and species identification conducted based on analysisof the DNA sequence (i.e., using BLAST).

The invention also features a method of using Candida specific primers(e.g., one or more of the DNA primers of SEQ ID NOs: 32 to 41) to detecta Candida species in a biological sample. The method may include use ofthe primers one or more of the primers for DNA sequencing. In severalembodiments, the primer is a Candida krusei primer having the sequenceof SEQ ID NO: 32 or SEQ ID NO: 33; the primer is a Candida albicansprimer having the sequence of SEQ ID NO: 34 or SEQ ID NO: 35; the primeris a Candida glabrata primer having the sequence of SEQ ID NO: 36 or SEQID NO: 37; the primer is a Candida parapsilosis primer having thesequence of SEQ ID NO: 38 or SEQ ID NO: 39; or the primer is a Candidatropicalis primer having the sequence of SEQ ID NO: 40 or SEQ ID NO: 41.

The invention also features fluorescence based methods of using thenucleic acid probes and primers described above (e.g., the nucleic acidmolecules of any one or more of SEQ ID NOs: 1 to 41) to detect Candidaspecies in a biological sample. These methods include amplifying andsequencing target nucleic acid molecules from Candida species in abiological sample. In several embodiments, the methods include, e.g., aTaqMan probe based assay (e.g., real-time or end-point PCR based TaqManassay using probes having sequence of SEQ ID NOs: 1 to 5);strand-displacement probe based assays (e.g., using probes havingsequence of SEQ ID NOs: 1 to 10); PCR assays using DNA binding dyes(e.g., real-time PCR based assay using SYBR® green dye and probes havingsequence of SEQ ID NOs: 1 to 41); and in situ hybridization assays usingfluorescently labeled species-specific probes (e.g., using probes havingsequence of SEQ ID NOs 1 to 41). In several embodiments, the nucleicacid probes having the sequence of any one of SEQ ID NOs: 6-10, and13-31 may have a detection label (e.g., a fluorescent label), such thatthe fluorescence detection is via a secondary step (e.g., a digoxigeninlabel for antibody-based detection using fluorescently labeledanti-digoxigenin antibodies, or a biotin label for detection usingfluorescent streptavdin). Alternatively the detection readout may benon-fluorescent. For example, nucleic acid probes having the sequence ofany one of SEQ ID NOs: 6-10 and 13-31 may have non-fluorescent detectionlabels, such as a radioactive isotope for autoradiographic detection,digoxigenin for antibody-based detection using a horse-radish peroxidase(HRP) conjugated anti-digoxigenin antibody, and biotin for streptavidinbased detection using HRP conjugated straptividin.

In other embodiments, the fluorescence based methods for detecting thepresence of at least one Candida species in a biological sample includesthe use of molecular beacon probes (e.g., those probes having thesequences of any one of SEQ ID NOs: 1 to 41, and particularly SEQ IDNOs: 1 to 5) having a fluorescent label, which may be the same ordifferent. Following contacting of the probe to the sample that includesnucleic acid molecules from the at least one Candida species andhybridization of the probe to the nucleic acid molecules, the presenceof the at least one Candida species is detected by heating the samplethrough a melting temperature (Tm) of at least one of the probes toobtain a melting curve. A decrease in fluorescence of the sample at theTm of at least one the probes indicates the presence of the at least oneCandida species. Preferably, the molecular beacon probes used in themethods have melting temperatures that vary by at least 1° C., such thata separate melt curve can be obtained for each molecular beacon probe.In particular embodiments, e.g., in which the methods are performedusing the PCR conditions set for in Example 1, a decrease influorescence at a Tm of ˜62-67° C. (e.g., ˜64° C.) in the presence ofthe probe having the sequence of SEQ ID NO: 1 indicates the presence ofCandida albicans in the sample; a decrease in fluorescence at a Tm of˜55-61° C. (e.g., ˜58° C.) in the presence of the probe having thesequence of SEQ ID NO: 2 indicates the presence of Candida tropicalis inthe sample; a decrease in fluorescence at a Tm of ˜65-71° C. (e.g., ˜67°C.) in the presence of the probe having the sequence of SEQ ID NO: 3indicates the presence of Candida glabrata in the sample; a decrease influorescence at a Tm of ˜60-65° C. (e.g., ˜62° C.) in the presence ofthe probe having the sequence of SEQ ID NO: 4 indicates the presence ofCandida parapsilosis in the sample; and a decrease in fluorescence at aTm of ˜66-72° C. (e.g., ˜69° C.) in the presence of the probe having thesequence of SEQ ID NO: 5 indicates the presence of Candida krusei in thesample. Many conditions may affect the Tm of a probe during a meltingcurve analysis, e.g., the salt(s) and salt concentration(s) present inthe reaction, the presence of intercalating agents, and the pH of thereaction. Accordingly, the melting temperatures indicated above for SEQID NOs: 1-5 are not meant to be limiting and may differ depending uponthe conditions used in the method.

In yet other embodiments, one or more of the nucleic acid probes of theinvention (e.g., any one of the nucleic acid probes having the sequenceof SEQ ID NOs: 1 to 10 and 13 to 41) may be used in an array ormicroarray based platform for detecting one or more Candida species in abiological sample.

Each of the methods of the invention may further include steps forprocessing samples (e.g., biological samples), which may include one ormore of the following: mixing the sample with a lysis agent solution(e.g., a detergent or a hypotonic solution); centrifuging the sample toform a supernatant and a pellet; discarding some or all of thesupernatant; and/or resuspending the pellet to form an extractcontaining the fungal cells. In yet other embodiments, the processingsteps may further include one or more of the following: optionallywashing the pellet (e.g., with TE buffer) prior to resuspending thepellet; lysing fungal cells of the extract by chemical methods (e.g.,using any combination of enzymes, or detergents, or surfactants),mechanical methods (e.g., using beads such as glass beads, bead beating,use of a finned tube in combination with beads, using beads in anagitation mill, use of beads with a chelating agent, use of glassshards, use of solid particles, use of beads or solid particles withmechanical or magnetic vortex centrifugation use of ultrasound, and/oruse of sonication), or methods involving temperature changes (e.g., useof heat (e.g., a temperature in the range of about 85° C. to about 125°C., such as a temperature of about 95° C.), use of freeze-thaw, and/oruse of freeze-boil) to form a lysate; and/or placing the lysate in adetection tube. Other processing steps may optionally include amplifyingnucleic acids present in a processed sample to form an amplified lysatesolution; adding all or a portion of the amplied lysate solution to adetection tube that includes one or more Candida specific nucleic acidprobes or primers and allowing contact between the target nucleic acidmolecules in the lysate and the one or more Candida specific nucleicacid probes or primers under conditions that allow hybridization of thenucleic acid probe(s) with the target nucleic acid molecules; anddetecting hybridization of the nucleic acid probe(s) or primers to theCandida target nucleic acid molecules whereby the detection of bindingof the Candida species-specific probe(s) with Candida target nucleicacid molecules present in the biological sample indicates the presenceof the Candida species in the biological sample.

In another embodiment, the amplified lysate solution described above maybe further processed to isolate the Candida nucleic acid molecules,e.g., by using an ion exchange column (such as a Qiagen column), glassor silica-base reverse phase adsorption/desorption, SPRI technology,phenol-chloroform extraction and ethanol precipitation, and/or the CTABmethod.

In several embodiments, each of the methods of the invention may furtherinclude an amplification step for increasing the amount of the targetCandida nucleic acid molecules to be detected in the biological sample.The amplification may include enzymatic amplification by, e.g., a PCRreaction. Primers having the sequence of SEQ ID NOs: 11 and 12 may beused as pan-Candida universal forward and reverse primers, respectively,to produce a Candida amplicon. In other embodiments, primers having thesequences of SEQ ID NOs: 6 to 41 can be used in combination with thepan-Candida universal primers to produce Candida species amplicons. Theamplification may be symmetric to produce a double stranded amplicon orthe amplification may be asymmetric to produce a single strandedamplicon.

In several embodiments, each of the methods of the invention fordetecting at least one Candida species in a biological sample can becompleted within at least about 5 hours or less (e.g., within 4.0 hours,3.5 hours, 3.0 hours, 2.5 hours, 2 hours, 1.5 hours, or 1 hour or less).In still other embodiments, each of the methods of the invention can beused to detect at least one Candida species in a sample when present ata concentration of at least about 4 Candida cells/mL (e.g., 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 Candida cells/mL).

In another embodiment of the compositions and methods of the invention,the biological sample is, e.g., a soil, water, or food sample or atissue or fluid sample from an organism, such as a human (e.g., wholeblood, sweat, tears, urine, saliva, semen, serum, plasma, cerebrospinalfluid (CSF), feces, vaginal fluid or tissue, sputum, nasopharyngealaspirate or swab, lacrimal fluid, mucous, or epithelial swab (buccalswab), tissues, organs, bone, teeth, and tumors).

In another embodiment, the invention features a kit which includes atleast one nucleic acid probe or primer having a sequence selected fromany of SEQ ID NOs: 1 to 41 and further includes one or more reagents forattachment of fluorescent label or magnetic nanoparticle to the probe,and/or one or more reagents for sample lysis, fungal lysis, isolation ofCandida nucleic acid, detection of Candida species, and nucleic acidamplification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are schematics showing binding of molecular beaconprobes to a target nucleic acid molecule. FIG. 1A shows the binding of aconventional molecular beacon probe. FIG. 1B shows the binding of ashared-stem molecular beacon probe of the present invention. Ashared-stem molecular beacon probe is designed so that 1) a stem-loophairpin is formed by the 5′ and 3′ ends of the probe, 2) a fluorophoreand quencher are attached to the 5′ and 3′ ends and sufficient quenchingwill occur when the hairpin structure is formed, 3) the probes have a“loop” sequence that is specific for the target Candida sequence to beidentified in the assay, and 4) sufficient fluorophore signal may bedetected upon hybridization of the molecular beacon probe to thespecific target sequence of Candida. The molecular beacon probes aredesigned to have a melting temperature (Tm) that is about 5 to 10degrees (e.g., ˜7 degrees) higher than the annealing temperature ofamplification primers that may be present with the molecular beaconprobes in an assay sample, and the molecular beacon probes arethermodynamically characterized to form no self dimers or heterodimerswith other probes or primers (e.g., pan-Candida universal PCR primers)that may be present in an assay sample.

FIGS. 2A and 2B are graphs showing detection of Candida krusei inreal-time PCR reactions. FIG. 2A shows the detection of Candida kruseiin a sample using purified genomic DNA. FIG. 2B shows the detection ofCandida krusei in a sample prepared from a cell lysate.

FIGS. 3A-3F are graphs showing titration curves for detection of Candidaspecies in real-time PCR reactions. FIGS. 3A-3C are titration curvesshowing detection of Candida parapsilosis (FIG. 3A), Candida tropicalis(FIG. 3B), and Candida krusei (FIG. 3C) using various amounts ofpurified genomic DNA. FIGS. 3D-3F are titration curves showing detectionof Candida parapsilosis (FIG. 3D), Candida tropicalis (FIG. 3E), andCandida krusei (FIG. 3F) using a cell lysate.

FIGS. 4A-4D are graphs showing titration curves for detection of Candidaalbicans and Candida glabrata in real-time PCR reactions. FIGS. 4A and4B are titration curves showing detection of Candida albicans (FIG. 4A)and Candida glabrata (FIG. 4B) using various amounts of purified genomicDNA. FIGS. 4C and 4D are titration curves showing detection of Candidaalbicans (FIG. 4C) and Candida glabrata (FIG. 4D) using a cell lysate.

FIGS. 5A-5D are graphs showing the use of multiple molecular beaconprobes in a multiplex assay to detect one or more Candida species in asample. In this case, the genomic DNA of Candida parapsilosis andCandida tropicalis are present in a single reaction and amplified in thepresence of a single beacon (FIGS. 5A and 5C, SEQ ID NO: 4 and SEQ IDNO: 2, respectively labeled with FAM and HEX) or with both beaconswithin the same reaction and emissions are observed in parallel usingtwo channels (FIG. 5B and FIG. 5D).

FIG. 6 is a schematic showing alignment of Candida amplicon sequencesthat are generated by amplification using the pan-Candida primers. Thealignment shows species-specific differences within this amplicon. Shownare sequences for C. krusei (pan fungal amplicon; SEQ ID NO. 42), C.albicans (pan fungal amplicon; SEQ ID NO: 43), C. glabrata (pan fungalamplicon; SEQ ID NO: 44), C. parapsilosis (pan candida amplicon; SEQ IDNO. 45), C. tropicalis (pan candida amplicon; SEQ ID NO: 46), C.parapsilosis (pan candida amplicon, SEQ ID NO: 47), C. tropicalis (pancandida amplicon, SEQ ID NO: 48), C. lusitaniae (ITS2; SEQ ID NO: 49),C. guillermondi (amplicon 2; SEQ ID NO: 50), and a majority sequence(SEQ ID NO: 51).

FIG. 7 is a sequence chromatogram generated from amplification withinwhole blood lysate using the pan-Candida forward and reverse primers(SEQ ID NOs: 11 and 12), followed by a phenol/chloroform extraction, achloroform extraction, and then subsequentSanger Dideoxy terminatorsequence analysis using Big Dye Vs.31. (Applied Biosystems, Foster City,Calif.).

FIGS. 8A-8E are graphs of titration curves showing detection of Candidaalbicans (FIG. 8A), Candida glabrata (FIG. 8B), Candida krusei (FIG.8C), Candida parapsilosis (FIG. 8D), and Candida tropicalis (FIG. 8E) inreal-time PCR reactions using genomic DNA obtained from Candida celllysates prepared with cells in an amount ranging from ˜520 cells to ˜3cells/mL (see the data in Tables 20, 22, 24, 26, and 28, which was usedto produce the graphs of FIGS. 8A-8E, respectively).

FIGS. 9A and 9B are graphs showing nucleic acid denaturation melt curvesin a multiplex reaction that includes Candida species-specific nucleicacid molecules from, and Candida species-specific molecular beaconprobes for, C. albicans, C. glabrata, C. parapsilosis, and C. tropicalis(FIG. 9A) and for C. albicans and C. krusei (FIG. 9B). TheCandida-specific molecular beacon probes used in the multiplex reactionsare all labeled with a HEX fluorophore. The presence of nucleic acidmolecules for each Candida species is determined by detecting a decreasein fluorescence as the sample is heated through the melting temperature(Tm) of each Candida species-specific molecular beacon probe. A decreasein fluorescence is observed only when hybridized beacon probes melt offtheir target nucleic acid molecules and the step-loop structure of theprobes reforms. No melt curve is observed when the probe-specific targetnucleic acid molecules are not present in the reaction.

DETAILED DESCRIPTION OF THE INVENTION

The invention features compositions, methods, and kits for detecting atleast one Candida species in a variety of media and biological samples,including, for example, biofluids, tissue samples, culture samples(e.g., a blood culture), food products, water samples, and soil samples.For example, the biological sample may include samples which have beenprocessed to remove patient tissue and/or cellular debris or samplesthat are substantially unprocessed, such as a whole blood sample. Thebiological sample may further include cell suspensions or lysates thatare produced by the methods of the invention. The biological sample mayfurther include any sample which contains at least one nucleic acidmolecule of Candida species; or any sample in which the nucleic acidmolecules of Candida species have been substantially isolated, enrichedor purified; or any sample in which the nucleic acid molecules ofCandida species have been amplified and enriched as an amplicon.

The methods of the invention for detecting a Candida species can beperformed with little to no sample preparation. In addition, the methodsmay be performed as a singleplexed assay to detect a single Candidaspecies or as a multiplexed assay that allows for detection of multipleCandida species in a single sample. The methods of the invention allowfor rapid and accurate detection of Candida in a biological sample(e.g., a tissue or fluid sample from a patient), which may be used tofacilitate point-of-care clinical decision making. The methods describedherein can be used to provide clinically and epidemiologicallymeaningful targeting of anti-fungal drug therapy to patients in needthereof and to provide optimal therapeutic outcome.

The compositions of the invention for use in the detection of a Candidaspecies (e.g., one or more of Candida albicans, Candida tropicalis,Candida krusei, Candida glabrata, Candida parapsilosis, Candidadubliniensis, Candida lusitaniae, and Candida guillermondi) in abiological sample include probes (e.g., molecular beacon probes and NMRassay probes) that can be used to detect nucleic acid molecules of theCandida species (e.g., in a real-time probe-based nucleic acid detectionassay). The methods of the invention involve contacting a sample thatincludes a nucleic acid molecule of at least one Candida species with atleast one probe that is capable of hybridizing to the nucleic acidmolecule and detecting hybridization between the probe and the nucleicacid molecule. In multiplexed assays, the sample may be contacted withtwo or more probes (e.g., probes to any two, three, four, or five ormore of the Candida species listed above) in order to detecthybridization between the probes and the nucleic acid molecules.

The methods of the invention may be fluorescence based detection methodsusing fluorescently labeled species-specific probes (e.g., molecularbeacon probes), such that hybridization between the probe and thenucleic acid molecule of the Candida species may result in a fluorescentsignal that can then detected by an appropriate detection device (e.g.,a real-time thermal cycler or other fluorescence detection devices knownin the art). For example, the probes may be fluorescently labeledmolecular beacon probes designed to hybridize to Candidaspecies-specific target nucleic acid molecules for detection of specificCandida species in a biological sample. Each Candida species-specificprobe may be labeled with a fluorescent probe that distinguishes thatprobe from the other Candida species-specific probe(s) (e.g., based ondifferences in the excitation or emission spectra of the differentprobes). Alternatively, the probes may all include the same fluorescentlabel, in which case distinguishing the hybridization of each Candidaspecies-specific probe to its targets is determined by other techniques,such as differences between the melting temperature (Tm) of each Candidaspecies-specific probe, as is discussed below.

The methods of the invention may further include amplifying nucleic acidmolecules of the Candida species present in the biological sample priorto detection of the Candida nucleic acid molecules. Amplification can beused to increase the amount of the Candida nucleic acid moleculespresent in the biological sample in case the original amount of Candidatarget nucleic acid molecules in the biological sample is below thedetection limit of the probe(s). Typically such amplification may bedone using one or more amplification primers. Any nucleic acidamplification method known in the art (e.g., polymerase chain reaction(PCR)) may be used. Preferably, the amplification method includes theuse of primers that amplify nucleic acid molecules of at least oneCandida species present in the biological sample.

Other methods of the invention include the use of fluorescently labeledprobes and/or primers of the invention for detection of at least oneCandida species in a biological sample in fluorescence based detectionassays, for example, in situ hybridization assays. Another detectionmethod employs use of a double strand specific intercalating dye, suchas SYBR® (Molecular Probes, Eugene, Oreg.). The fluorescence basedassays may be performed in conjunction with real-time PCR detection. Theinvention also features one or more probes and primers of the inventionfor use as TaqMan probes in a real-time PCR assay or an end-point PCRassay. Finally, the Candida specific primers of the invention (e.g., anyone or more of SEQ ID NOs: 1 to 10 and 13 to 41) may be used insequencing based assays or in array or microarray based platforms inorder to detect the presence of at least one Candida species in abiological sample.

The methods of the invention also include NMR based assays using Candidaspecies-specific probes conjugated to magnetic nanoparticles. Forexample, in such an assay a sample may be contacted with at least oneCandida species-specific probe that is conjugated to a magneticnanoparticle, such that hybridization between the probe and the nucleicacid molecule of the Candida species may produce an NMR signal that maybe detected by NMR spectroscopy. Other aspects of the compositions,methods, and kits of the invention are described below.

Compositions and Methods for Detecting Candida Species in BiologicalSamples.

The invention features compositions, methods, and kits for detecting atleast one Candida species in a biological sample. The invention featuresCandida species-specific nucleic acid probes having the sequence of anyone of SEQ ID NOs: 1 to 10 and 13 to 31, and probes having at least 90%,preferably at least 95%, more preferably at least 97%, and mostpreferably at least 99% or more identity to these sequences, that may beemployed in a number of assays to detect one or more (for e.g., 2, 3, 4,5, 6, 7, 8, or more) Candida species in a biological sample. Forexample, the probes may be used to detect one or more of Candidaalbicans, Candida tropicalis, Candida krusei, Candida glabrata, Candidaparapsilosis, Candida dubliniensis, Candida lusitaniae, and Candidaguillermondi, in any combination, in a biological sample.

The methods of the invention include the use of one or more Candidaspecific probes and/or primers for detecting the presence of a Candidatarget nucleic acid molecule present in a biological sample. The methodsinclude one or more of the following steps: (a) providing a biologicalsample; (b) mixing the sample with a lysis agent solution; (c)centrifuging the sample to form a supernatant and a pellet; (d)discarding some or all of the supernatant; and (e) resuspending thepellet to form an extract containing the fungal cells, optionallywashing the pellet (e.g., with TE buffer) prior to resuspending thepellet and optionally repeating step (c); (f) lysing cells of theextract by chemical or mechanical methods to form a lysate; (g) placingthe lysate of step (f) in a detection tube; (h) optionally amplifyingnucleic acids therein to form an amplified lysate solution; (i) addingto the detection tube one or more Candida specific nucleic acid probesand contacting the target nucleic acid molecules in the lysate with oneor more Candida specific nucleic acid probes under conditions that allowhybridization of the probe(s) (also referred to as binding of theprobe(s)) to the target nucleic acid molecules, and (j) detectinghybridization of the probe(s) to the Candida target nucleic acidmolecules by methods described below. The detection of binding of theCandida species-specific probe(s) with Candida target nucleic acidmolecules present in the biological sample indicates the presence of theCandida species in the biological sample. The methods of the inventionprovide rapid and accurate readouts of the presence of at least onespecies of Candida in the sample. Two or more probes of the inventionmay be used in the methods of the invention to perform a multiplexingassay that provides for the detection of two or more Candida species(e.g., 2, 3, 4, or 5 or more of the Candida species described herein) inthe biological sample.

In certain embodiments, steps (a) through (h) are completed within atleast about 5 hours or less (e.g., within 4.0 hours, 3.5 hours, 3.0hours, 2.5 hours, 2 hours, 1.5 hours, or 1 hour or less). In particularembodiments, the methods allow for (i) at least 95% correct detection atless than or equal to 5 Candida cells/mL in samples spiked into 50individual healthy patient blood samples; (ii) at least 95% correctdetection at less than or equal to 5 Candida cells/mL in samples spikedinto 50 individual unhealthy patient blood samples; (iii) greater thanor equal to 80% correct detection in clinically positive patient samples(e.g., Candida positive by another technique, such as by cell culture)starting with 2 mL of biological sample; and/or (iv) at least 90%correct detection in biological samples containing at least 5 Candidacells/mL. The invention provides methods in which Candida species can bedetected at pathogen concentration of, e.g., at least 3 Candida cells/mL(e.g., 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 Candidacells/mL) in the sample with a coefficient of variation of less than 15%(e.g., 10 cells/mL with a coefficient of variation of less than 15%,10%, 7.5%, or 5%; or 25 cells/mL with a coefficient of variation of lessthan 15%, 10%, 7.5%, or 5%; or 50 cells/mL with a coefficient ofvariation of less than 15%, 10%, 7.5%, or 5%; or 100 cells/mL with acoefficient of variation of less than 15%, 10%, 7.5%, or 5%). Themethods of the invention may be used to detect the presence of Candidain a biological sample having a volume in the range of, e.g., 0.05 to10.0 mL (e.g., a biological sample having a volume in the range of 0.05to 0.25 mL, 0.25 to 0.5 mL, 0.25 to 0.75 mL, 0.4 to 0.8 mL, 0.5 to 0.75mL, 0.6 to 0.9 mL, 0.65 to 1.25 mL, 1.25 to 2.5 mL, 2.5 to 3.5 mL, 3.0to 4.0 mL, or 3.0 to 10 mL).

Species-Specific Candida Probes of the Invention

Compositions of the invention include Candida species-specific nucleicacid probes that can bind to target sequences within the endogenousnucleic acid sequences of specific Candida species. The nucleic acidprobes of the invention are designed to hybridize to endogenous nucleicacid molecules of Candida species and to provide accurate detection ofat least one Candida species in a biological sample tested. The nucleicacid probes can be used individually in a detection assay or two or morenucleic acid probes (e.g., 3, 4, or 5 or more probes) can be used incombination, e.g., in a multiplex fluorescence assay, or in an NMRaggregation assay, to detect one or more Candida species in thebiological sample.

The nucleic acid probes of the invention include: 5′-GGT CAA AGT TTG AAGATA TAC GTG GTT GAC C-3′ (SEQ ID NO: 1) for detecting Candida albicans,5′-CTA GCA AAA TAA GCG TTT TTG GAT GCT AG-3′ (SEQ ID NO: 2) fordetecting Candida tropicalis, 5′-CAG CAC GCA CAA AAC ACT CAC TTA TTGCTG-3′ (SEQ ID NO: 3) for detecting Candida glabrata, 5′-GTC GAA TTT GGAAGA AGT TTT GGT TTC GAC-3′ (SEQ ID NO: 4) for detecting Candidaparapsilosis, 5′-CCT GAT TTG AGG TCG AGC TTT TTG TAT CAG G-3′ (SEQ IDNO: 5) for detecting Candida krusei, 5′-GGT CAA AGT TTG AAG ATA TAC GTGG-3′ (SEQ ID NO: 6) for detecting Candida albicans, 5′-CTA GCA AAA TAAGCG TTT TTG GA-3′ (SEQ ID NO: 7) for detecting Candida tropicalis,5′-CAG CAC GCA CAA AAC ACT CAC TTA T-3′ (SEQ ID NO: 8) for detectingCandida glabrata, 5′-GTC GAA TTT GGA AGA AGT TTT GGT-3′ (SEQ ID NO: 9),for detecting Candida parapsilosis, 5′-CCT GAT TTG AGG TCG AGC TTT TTGT-3′ (SEQ ID NO: 10) for detecting Candida krusei,5′-AATAAAATGGGCGACGCCAGAGACCGCCTT-3′(SEQ ID NO: 26) and5′-GCATCTCCGCCTTATACCACTATCA-3′ (SEQ ID NO: 27) for detecting Candidadubliniensis, 5′-GGTTGATATTCGGAGCAACGCC-3′ (SEQ ID NO: 28) and5′-GTCCTACCTGATTTGAGGGCGAAAT-3′ (SEQ ID NO: 29) for detecting Candidalusitaniae, 5′-GCAAACGCCTAGTCCGACTAAGAGTATCACTCAATACC-3′ (SEQ ID NO: 30)and 5′-TGTAAGGCCGGGCCAACAATACCAGAAATATCCCGC-3′ (SEQ ID NO: 31) fordetecting Candida guillermondi, 5′-CCGAGAGCGAGTGTTGCGAGA-3′ (SEQ ID NO:32) and 5′-TCTCGCAACACTCGCTCTCGG-3′ (SEQ ID NO: 33) for detectingCandida krusei, 5′-GGTAACGTCCACCACGTATATCT-3′ (SEQ ID NO: 34) and5′-AGATATACGTGGTGGACGTTACC-3′ (SEQ ID NO: 35) for detecting Candidaalbicans, 5′-GGGAGGGATAAGTGAGTGTGTGCGT-3′ (SEQ ID NO: 36) and5′-ACGCACAAAACACTCACTTATCCCTCCC-3′ (SEQ ID NO: 37) for detecting Candidaglabrata, 5′-GGTACAAACTCCAAAACTTCTTCC-3′ (SEQ ID NO: 38) and5′-GGAAGAAGTTTGGAGTTTGTACC-3′ (SEQ ID NO: 39) for detecting Candidaparapsilosis, and 5′-GCTAGTGGCCACCACAATITATITCA-3′ (SEQ ID NO: 40) and5′-TGAAATAAATTGTGTGGCCACTAGC-3′ (SEQ ID NO: 41) for detecting Candidatropicalis. The invention features the probes described above, as wellas probes having at least 90%, 95%, 97%, 99%, or more sequence identityto these probes.

In a further embodiment, one or more of the probes described above maybe used in combination with any one of the Candida species-specificprobes having the sequences of SEQ ID NOs: 13 to 25, which are describedin International Application Nos. PCT/US2011/56933 and PCT/US2011/56936,both of which are incorporated herein by reference. These probesinclude: 5′-ACC CAG COG TTT GAG GGA GAA AC-3′ (SEQ ID NO: 13) and 5′-AAAGTT TGA AGA TAT ACG TGG TGG ACG TTA-3′ (SEQ ID NO: 14) for detectingCandida albicans; 5′-CGC ACG CGC AAG ATG GAA ACG-3′ (SEQ ID NO: 15),5′-AAG TTC AGC GGG TAT TCC TAC CT-3′ (SEQ ID NO: 16) and 5′-AGC TTT TTGTTG TCT CGC AAC ACT CGC-3′ (SEQ ID NO: 17) for detecting Candida krusei;5′-CTA CCA AAC ACA ATG TGT TG AGA AG-3′ (SEQ ID No: 18) and 5′-CCT GATTTG AGG TCA AAC TTA AAG ACG TCT 0-3′ (SEQ ID NO: 19) for detectingCandida glabrata; 5′-AGT CCT ACC TGA TTT GAG GTCNitIndAA-3′ (SEQ ID NO:20), 5′-CCG NitIndGG OTT TGA GGG AGA AAT-3′ (SEQ ID NO: 21), 5′-AAA GTTATG AAATAA ATT GTG GTG GCC ACT AGC-3′ (SEQ ID NO: 22), 5′-ACC CGG GGGTTTGAG GGA GAA A-3′ (SEQ ID NO: 23), 5′-AGT CCT ACC TGA TTT GAG GTC GAA-3′(SEQ ID NO: 24), and 5′-CCG AGG GTT TGA GGG AGA AAT-3′ (SEQ ID NO: 25)for detecting either Candida parapsilosis or Candida tropicalis. Inother embodiments, reverse complement versions of one or more of thenucleic acid molecules of SEQ ID NOs 1 to 41 may also be used as probesfor detecting Candida species in a biological sample or as primers foramplying one or more Candida nucleic acid molecules in a biologicalsample in an amplification step.

In addition, Candida specific nucleic acid molecules with at least 90%sequence identity (or at least 90%, 95%, 97%, 99%, or more sequenceidentity) to the sequences of SEQ ID NOs: 1 to 41, or to the reversecomplement sequences of SEQ ID NOs: 1 to 41, may be used for detecting,amplifying, or both amplifying and detecting Candida species nucleicacid molecules in a biological sample.

The Candida species-specific probes of the invention may also include adetection label (e.g., a fluorescent detection label) or may beconjugated to a magnetic nanoparticle for use in the methods of theinvention for detecting at least one Candida species in a biologicalsample. Detection labels and magnetic nanoparticles for use in thepresent invention, and methods for using probes of the invention thatinclude a detection label or that are conjugated to a magneticnanoparticle, are described in detail below.

Composition and Methods for Fluorescence-Based Detection of CandidaSpecies in Biological Samples

The invention features Candida species-specific nucleic acid probes thatcan bind to target sequences within the endogenous nucleic acidsequences of Candida. These probes can be used to detect the presence ofspecific species of Candida in biological samples. The probes may belabeled with a fluorescent label (also referred to as a fluorophore) andapplied to a Candida lysate prepared from the biological sample.Preferably, the fluorophore-labeled probe emits a fluorescent signalupon hybridization of the probe to its target sequence and a fluorescentsignal is not produced by unbound probe, e.g., as described in U.S.Patent Application No. US 2010/0221710A1 (incorporated herein byreference). The fluorescent signal produced upon hybridization betweenprobe and target nucleic acid of the Candida species can be detected ina fluorescence detection device (e.g., a real time thermal cycler orother detection device known in the art). Each of the Candidaspecies-specific probes described herein may be differentially labeledwith fluorphores with spectrally distinguishable emission spectra foruse in a multiplex fluorescence detection assay. In such a multiplexassay, two or more fluorescently labeled nucleic acid probes (e.g., 3,4, or 5 or more probes), each having a different fluorophore attached,can be used in combination to rapidly and accurately detect multipledifferent Candida species in a biological sample.

The Candida species-specific probes of the present invention may also beused as “molecular beacons.” Methods to prepare and use molecularbeacons are fully described in, e.g., U.S. Pat. Nos. 5,118,801,5,312,728, and 5,925,517, each of which is incorporated herein byreference. Typically, molecular beacon probes have a fluorophoreattached at the 5′ end of the probe and a non-fluorescent quencherattached at the 3′ end of the probe. Molecular beacons further containcomplementary sequences at the 5′ and 3′ ends allowing these two ends tohybridize. The two ends flank a middle intervening “loop” sequence. The5′ and 3′ ends of the molecular beacon can hybridize to each other andform an intramolecular stem structure and the middle interveningsequence region forms a “loop.” The “loop” part of the molecular beaconcan hybridize to the target nucleic acid.

Under conditions (e.g., an appropriate temperature range) when the“loop” part of the probe cannot hybridize with the target nucleic acidsequence, the 5′ and 3′ ends of the probe form the intramolecular stemstructure. As a result, the proximity between the fluorophore andquencher causes the quencher to absorb any fluorescence emitted by thefluorophore and no fluorescence is produced. However, under differentconditions (e.g., an appropriate temperature range) that allow the“loop” part of the probe to hybridize to the target nucleic acidsequence, the stem structure is not formed, the fluorophore is separatedfrom the quencher and, as a result, a fluorescent signal is produced.

The molecular beacon probes of the present invention are designed todetect specific nucleic acid target sequences in Candida species viaspecies-specific “loop” sequences. Use of different fluorophore labelson the 5′ end of each of the species-specific probes can allow use ofmultiple molecular beacon probes in a multiplex assay to simultaneouslyand rapidly detect multiple Candida species in a biological sample.Alternatively, each of the Candida species-specific probes describedherein can be prepared as molecular beacon probes and labeled with thesame fluorphore or with fluorophores having overlapping emission spectrafor use in a multiplex fluorescence detection assay. Since each of theCandida species-specific molecular beacon probes have a differentmelting temperature (Tm), hybridization of each of the Candidaspecies-specific molecular beacon probes to their target nucleic acidmolecules can be detected by assaying fluorescence through a range oftemperatures that includes the Tm of each probe used.

For example, a sample containing nucleic acid molecules from a Candidaspecies may be contacted with a Candida albicans specific molecularbeacon probe having a Tm of ˜64° C. (e.g., a Tm of 62-67° C.) and a HEXfluorophore and a Candida glabrata specific molecule beacon probe havinga Tm of ˜67° C. (e.g., a Tm of 65-71° C.) and a HEX fluorophore.Fluorescence in the sample may be due to binding of the C. albicans orthe C. glabrata probes or both. To determine which probe(s) are bound totheir target nucleic acid molecules, the temperature of the sample maybe raised from, e.g., 40° C. to 80° C. while detecting changes influorescence in the sample. If C. albicans nucleic acid molecules arepresent in the sample, there will be a change in fluorescence as thesample temperature increases through 64° C. In this case, half of theCandida albicans-specific molecular beacon probes will dissociate fromtheir target nucleic acid molecules, which will result in a decrease influorescence as the probe melts off and the stem-loop structure reformsand quenches the fluorescence of those probes. If C. glabrata nucleicacid molecules are present in the sample, there will be a decrease influorescence as the temperature is increased through 67° C. In thiscase, half of the C. glabrata probes will dissociate from their targetnucleic acid molecules, which will result in a decrease in fluorescenceas the probe melts off and the stem-loop structure reforms and quenchesthe fluorescence of those probes. There would be no melt curves in theabsence of Candida nucleic acid molecules. Similarly, no change influorescence around 64° C. would indicate the absence of Candidaalbicans nucleic acid molecules, while no change in fluorescence around67° C. would indicate the absence of Candida glabrata.

The amount of Candida specific target nucleic acid molecules present ina biological sample may be sufficient for detection according to themethods of the present invention (e.g., by contact with thespecies-specific probes of the invention) without the need foramplification of the target nucleic acid molecules. However, it may bepreferable to increase the amount of target nucleic acid moleculespresent in the biological sample prior to detection (e.g., if the amountof target nucleic acid molecules present in the biological sample isbelow the detection limit of the probe(s)). In this case, an optionalamplification step may be performed to amplify the target nucleic acidmolecules of the Candida species, which will increase the amount of thetarget nucleic acid molecules present in the biological sample that maybe bound by the probe(s). Typically, such amplication may be performedprior to contact with the probe by using, e.g., PCR or any otheramplification methods known in the art.

The amplification reaction mixture may include, e.g., (1) the targetnucleic acid molecule(s) and (2) forward and/or reverse amplificationprimers specific for the target nucleic acid molecule(s). The forwardand/or reverse primers may include, for example, the sequence 5′-GGC ATGCCT GT TGA GCG TC-3′ (SEQ ID NO: 11) and the sequence 5′-GCT TAT TGA TATGCT TAA GTT CAG CGG GT-3′ (SEQ ID NO: 12), each of which is universal tomultiple Candida species, as described in International Application Nos.PCT/US2011/56933 and PCT/US2011/56936. These primers are referred toherein as “pan-Candida primers.” Alternatively, the forward and/orreverse primers can include primers that recognize species-specifictarget nucleic acids and can have the sequence of any one of SEQ ID NOs:6 to 10 and 13 to 25. The amplification produces a Candida amplicon inthe reaction mixture. The amplification may include substantially moreforward or reverse primers if the amplification is to be asymmetric, orboth a forward and a reverse primer in equimolar ratios, if theamplification is to be symmetric, as described in detail below.

In a further embodiment, the detection probe(s) may be added at any timeduring the amplification step. For example, a fluorescently labeledmolecular beacon probe (or probes in a multiplex assay) may be added toa sample prior to amplification (e.g., prior to, or substantiallysimultaneously with, addition of the amplification primer(s)) so thatduring the amplification step the probe binds to its target and afluorescent signal can be detected in real time. A real-time PCR machinemay used in such an assay and allows continuous monitoring of thefluorescence every time the species-specific probe hybridizes with itstarget nucleic acid sequence. Alternatively, a fluorescently labeledmolecular beacon probe (or probes in a multiplex assay) can be added toa sample after an amplification step, and under conditions that allowthe probe to hybridize with its target Candida amplicon, wherebyhybridization of the probe to an amplicon of a Candida target nucleicacid molecule can be detected by recording the resulting fluorescentsignal.

The detection device for detecting a fluorescent signal may include anydevice that can provide temperature conditions for probe hybridizationand that can detect a fluorescent readout when the hybridization occurs.For example, such a device may include a thermal cycler (e.g., areal-time thermal cycler). Alternatively, the device can be afluorescent plate reader, a flow-cytometer detecting particles withconjugated capture probes, a fluorescence microscope, or amicroarray-based capture/detection, each with the ability to maintainthe samples at a specific temperature range and the ability to detectand record fluorescent signals.

Fluorescently Labeled Species-Specific Candida Probes

Nucleic acid probes of the invention having the sequence of any one ofSEQ ID NOs: 1 to 5 (or probes having at least 90%, 95%, 97%, or 99% ormore sequence identity to the sequence of SEQ ID NOs: 1 to 5) may beused in an assay to detect at least one Candida species in a biologicalsample. Preferably, such probes include a detection label, such as afluorophore. The fluorophore can be conjugated to the 5′ end or the3′end of the probe, or alternatively it can be attached internallywithin the probe. Fluorophores that are known in the art and can be usedfor conjugation to probes of the invention, as well as the differenttypes of devices that may be used to detect such probes (e.g., differenttypes of thermal cyclers), are described in, e.g., Table 2 of Marras(Methods Mol. Biol. 335:3-16, 2006 (incorporated herein by reference)).Preferred examples of a fluorophore for labeling of the probes of theinvention include, for example, FAM, TAMRA, HEX, TMR, Cy3, and Cy5. Formultiplexing assays utilizing multiple probes for the simultaneousdetection of multiple Candida species, it may be preferable to attachfluorophores with spectrally distinguishable emission spectra onto eachspecies-specific probe in order to discriminate between binding of eachprobe to its respective target. Alternatively, as described above;multiplex assays may also be performed using multiple different Candidaspecies specific probes that are labeled with the same fluorophore.Fluorescently labeled probes may include, without limitation, TaqManprobes (also known in the art as 5′ nuclease probes),strand-displacement probes, or molecular beacon probes.

The fluorescently labeled probes of the invention having sequences ofSEQ ID NOS: 1 to 5 (or probes having at least 90%, 95%, 97%, or 99% ormore sequence identity to the sequence of SEQ ID NOs: 1 to 5) can bemolecular beacon probes that may further include a quencher attached tothe probe in addition to the fluophore, as described above. For example,the fluorophore can be attached to the 5′ end of the probe and thequencher can be attached to the 3′ end of the probe or vice versa. Thechoice of the specific quencher depends on the specific fluorophore thatis attached to the probe. Examples of quenchers that can be conjugatedto the molecular beacon probes of the invention include, for example,DDQ-I, Dabcyl, Eclipse, Iowa Black FQ, BHQ-1, QSY-7, BHQ-2, DDQ-II, IowaBlack RQ, QSY-21, BHQ-3. Examples of preferred fluorescent-quencherpairs used in the art are described in, e.g., Marras (supra; see Table 2therein), and include, without limitations, FAM-BHQ-1, TET-BHQ-1,HEX-BHQ-1, FAM-Dabcyl, TET-Dabcyl, and HEX-Dabcyl.

Nucleic acid probes of the invention having the sequence of SEQ ID NOs:1 to 5 (or probes having at least 90%, 95%, 97%, or 99% or more sequenceidentity to the sequence of SEQ ID NOs: 1 to 5) may be conventionalmolecular beacon probes (see FIG. 1A) or “shared-stem” molecular beaconprobes (see FIG. 1B). Shared-stem probes have one or more of thefollowing characteristics:

1) About five to ten nucleotides (for e.g., 4, 5, 6, 7, 8, or 9nucleotides; preferably 5 or 6 nucleotides) at the 5′ end that arecomplementary to about five to ten nucleotides (for e.g., 4, 5, 6, 7, 8,or 9 nucleotides; preferably 5 or 6 nucleotides) at the 3′ end such thatthe 5′ and 3′ ends of the probe can hybridize to form a “stem”structure;

2) A species-specific sequence of about 17 to 30 nucleotides in length(for e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,or 30 nucleotides) that resides in the middle “loop” region of theprobe, that has limited secondary structure, and that can bind tospecific target sequences in the endogenous nucleic acid of each Candidaspecies;

3) A shared-stem design such that the 5′ end of the nucleic acid probehas the ability to hybridize to a target sequence within the endogenousnucleic acid of each Candida species, whereas about 1 to 20 nucleotides(e.g., about 5-6 nucleotides) of the 3′ stem region do not hybridizewith the endogenous nucleic acid of Candida; and

4) A predicted stem Tm of ˜50° C. and a probe/target duplex Tm ofbetween 50° C. and 75° C. (e.g., ˜60° C.).

When the probe is a molecular beacon probe that includes a fluorophoreand a quencher, the shared-stem feature of the probe improves signal tobackground ratios, improves sensitivity due to increased stability ofthe molecular beacon probe-target duplex, potentially enhancesfluorogenic signals, and reduces the secondary structure in the probebackbone.

Each of the species-specific molecular beacon probes may be labeled witha different fluorophore (e.g., FAM, TAMRA, HEX, TMR, Cy3 and Cy5), suchthat the molecular beacon probes may be applied simultaneously to asingle sample in a multiplex assay that allows simultaneous detection ofmultiple Candida species based on spectral resolution of the emissionmaxima for the different fluorescent labels. The molecular beacon probesare designed to minimize heterodimer formation between the beacons sothe detection is amenable to multiplexing using different fluorophores,as described above.

Probes of the invention having the sequence of SEQ ID NOs: 1 to 5 (orprobes having at least 90%, 95%, 97%, or 99% or more sequence identityto the sequence of SEQ ID NOs: 1 to 5) may be used in combination withan optional PCR based amplification step. The amplification step, whenperformed in the methods of the invention, may be symmetric orasymmetric PCR. Symmetric PCR can be performed using the pan-Candidauniversal primers as the forward and reverse primers, which can amplifya Candida amplicon to increase the amount of all Candida species targetnucleic acid molecules present in the biological sample. Symmetric PCRmay also utilize the pan-Candida universal forward primer in combinationwith the complement of one or more of SEQ ID NOs: 6 to 10 and 13 to 25,or the pan-Candida universal reverse primer in combination with the oneor more of SEQ ID NOs: 6 to 10 and 13 to 25. Asymmetric PCR can beperformed using the pan-Candida forward or reverse primer, one or moreof SEQ ID NOs: 6 to 10 and 13 to 25 as the forward primer, or one ormore of the complement of SEQ ID NOs: 6 to 10 and 13 to 25 as thereverse primer.

For example, Candida specific target nucleic acid molecules preparedfrom a suspension of lysed Candida cells can be added to a tubecontaining an asymmetric PCR master mix that includes the pan Candidaprimers and the molecular beacon probe(s). The reaction tube may then beplaced in a real-time thermal cycler machine programmed to performmultiple rounds of amplification of the target nucleic acid moleculescharacteristic of the Candida species present in the mix. This allowscontinuous monitoring of the fluorescence every time the probehybridizes with its target nucleic acid sequence.

The molecular beacon probes may be designed to have a Tm about 7 degrees(for e.g., 3, 4, 5, 6, 7, 8, 9, or 10 degrees) higher than the annealingtemperature of the primers and are thermodynamically characterized toform no self or heterodimers with the pan-Candida universal primers orthe primers of SEQ ID NOs: 6 to 10 and 13 to 25. The limit of detectionof these molecular beacon probes is about 3 cells/m (for e.g., at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50cells/mL).

The amplification reaction may also be performed using pairwisecombinations of one of the universal primers and a primer selected fromany one of SEQ ID NOs: 13 to 25. The concentration of one of the primersmay be in excess compared to the other for asymmetric PCR. Such pairwisecombinations may be designed based-on the desired Candida speciesamplicon and the genomic position of the probe- and the primersequences. Such pairwise combinations may include, without limitation,the universal forward primer in combination with: a) a reverse primerhaving the reverse complement of primer of SEQ ID NO: 13 for amplifyinga Candida albicans amplicon, b) a reverse primer having the reversecomplement sequence of SEQ ID NOs: 20 to 25 for amplifying a Candidatropicalis or Candida parapsilosis amplicon, c) a reverse primer havingthe reverse complement sequence of SEQ ID NOs: 18 or 19 for amplifying aCandida glabrata amplicon, and d) a reverse primer having the reversecomplement sequence of SEQ ID NOs: 15 to 17 for amplifying a Candidakrusei amplicon.

Alternatively, the universal reverse primer may be used in combinationwith: a) a forward primer having the sequence of SEQ ID NO: 13 foramplifying a Candida albicans amplicon, b) a forward primer having thesequence of SEQ ID NOs: 20 to 25 for amplifying a Candida tropicalis orCandida parapsilosis amplicon, c) a forward primer having the sequenceof SEQ ID NOs: 18 or 19 for amplifying a Candida glabrata amplicon, andd) a forward primer having the sequence of SEQ ID NOs: 15 to 17 foramplifying a Candida krusei amplicon.

Alternate Methods for Detecting Candida Species Using Probes and Primersof the Invention

In a further embodiment of the invention, all probes and primersdescribed above can be used by methods known in the art for detecting,amplifying and sequencing target nucleic acid molecules from Candidaspecies in samples. For example, probes having the sequence of any oneof SEQ ID NOs: 6 to 10, and 13-41 can be used to detect one or moreCandida species in a biological sample in one or more of the followingmethods: a) a TaqMan probe based assay (e.g., real-time PCR based TaqManassay), b) strand-displacement probe based assays, c) PCR assays usingDNA binding dyes (e.g., real-time PCR based assay using SYBR® greendye), d) in situ hybridization assays using fluorescently labeledspecies-specific probes, e) sequencing based assays (e.g.,dideoxy-sequencing using probes having SEQ ID NOs: 32 to 41), and f)array or microarray based assays. Probes having the sequence of any oneof SEQ ID NOs: 6-10, and 13-41 may have detection labels such that thefluorescence detection is via a secondary step. In such cases, thedetection labels on the probe may include, without limitation,digoxigenin label for antibody-based detection using fluorescent labeledanti-digoxigenin antibodies, or biotin label for detecting usingfluorescent streptavdin. Probes having the sequence of any one of SEQ IDNOs: 6-10, and 13-41 may also have non-fluorescent detection labelsincluding, but not limited to, radioactive isotopes for autoradiographicdetection, digoxigenin for antibody-based detection using a horse-radishperoxidase (HRP) or alkaline phosphatase conjugated anti-digoxigeninantibody, and biotin for streptavidin based detection using HRP oralkaline phosphatase conjugated streptavidin. Non-fluorescent methodsfor detecting probe binding, such as autoradiographic detection, HRP- oralkaline phosphatase-based detection, colorimetric or chemiluminescentdetection, and detection using array or microarray platforms are wellknown in the art and can be adapted by those of skill in the art to themethods described herein for detecting Candida species in a biologicalsample.

Compositions and Methods for Detecting Candida Species in an NMR BasedAssay

An alternate method for detecting Candida species in a biological samplemay include the use of an NMR based assay using species-specific probesthat are designed to be compatible with such assays. Use of NMR basedassays to detect Candida species are described in, e.g., InternationalApplication No. PCT/US2011/56933 and International Application No.PCT/US2011/56936.

The present invention features probes having the sequence of any one ofSEQ ID NOs: 6 to 10 and 26 to 31 that are conjugated to magneticnanoparticles for use in an NMR based assay for detecting at least oneCandida species in a biological sample. These nucleic acid probes of theinvention include, e.g., 5′-GGT CAA AGT TTG AAG ATA TAC GTG G-3′ (SEQ IDNO: 6) for detecting Candida albicans, 5′-CTA GCA AAA TAA GCG TTT TTGGA-3′ (SEQ ID NO: 7) for detecting Candida tropicalis, 5′-CAG CAC GCACAA AAC ACT CAC TTA T-3′ (SEQ ID NO: 8) for detecting Candida glabrata,5′-GTC GAA TIT GGA AGA AGT TTT GGT-3′ (SEQ ID NO: 9), for detectingCandida parapsilosis, 5′-CCT GAT TTG AGG TCG AGC TTT TG T-3′ (SEQ ID NO:10) for detecting Candida krusei,5′-AATAAAATGGGCGACGCCAGAGACCGCCTT-3′(SEQ ID NO: 26) and5′-GCATCTCCGCCTTATACCACTATCA-3′ (SEQ ID NO: 27) for detecting Candidadubliniensis, 5′-GGTTGATATTTCGGAGCAACGCC-3′ (SEQ ID NO: 28) and5′-GTCCTACCTGATTTGAGGGCGAAAT-3′ (SEQ ID NO: 29) for detecting Candidalusitaniae, and 5′-GCAAACGCCTAGTCCGACTAAGAGTATCACTCAATACC-3′ (SEQ ID NO:30) and 5′-TGTAAGGCCGGGCCAACAATACCAGAAATATCCCGC-3′ (SEQ ID NO: 31) fordetecting Candida guillermondi. Probes having sequences with at least90%, e.g., at least 95%, 97%, and 99% or more sequence identity to thesequences of SEQ ID NOs: 6 to 10 and 26 to 31 and that are bound tonanoparticles are also included in the present invention.

As further described in International Application No. PCT/US2011/56933and International Application No. PCT/US2011/56936, the NMR assay may bean “aggregation assay” in which magnetic nanoparticles can include oneor more populations having a first probe and a second probe conjugatedto their surface, the first probe operative to bind to a first segmentof the target nucleic acid and the second probe operative to bind to asecond segment of the target nucleic acid. Upon probe hybridization tothe target nucleic acid, the magnetic particles form aggregates.

The terms “aggregation” and “aggregate” are used interchangeably in thecontext of the magnetic particles described herein and mean that two ormore magnetic particles are brought into close proximity to one anotheras a result of target nucleic aid molecule binding by thenanoparticle-conjugated probes. Hybridization between probe and targetnucleic acid may be detected in a nuclear magnetic resonance (NMR)instrument by measuring the “NMR relaxation rate,” as described inInternational Application No. PCT/US2011/56933 and InternationalApplication No. PCT/US2011/56936. NMR relaxation rate refers to ameasurement of any of the following in a sample: T₁, T₂, T₁/T₂ hybrid,T_(1rho), T_(2rho), and T₂*. The methods of the invention are designedto produce an NMR relaxation rate characteristic of whether a targetnucleic acid from Candida is present in a biological sample. In someinstances the NMR relaxation rate is characteristic of the quantity oftarget nucleic acid present in the sample. In addition to the“aggregation” assay described above, the probes can be used in“disaggregation” assays, according to methods described in InternationalApplication No. PCT/US2011/56933 and International Application No.PCT/US2011/56936.

The Candida lysate containing the target nucleic acid may be contactedwith magnetic nanoparticles conjugated to species specific probes havingthe sequence of any one or more of SEQ ID NOs: 6 to 10 and 26 to 31, ortheir complements, and used in combination with magnetic nanoparticlesconjugated with species-specific probes having the sequence of SEQ IDNOs: 13 to 25. For example, the probe of SEQ ID NO: 6 may be used incombination with a probe having the sequence of any one of SEQ ID NO: 13or 14 for detecting Candida albicans; the probe of SEQ ID NO: 7 may beused in combination with a probe having the sequence of any one of SEQID NOs: 20 to 25 for detecting Candida tropicalis; the probe of SEQ IDNO: 8 may be used in combination with a probe having the sequence of anyone of SEQ ID NO:18 or 19 for detecting Candida glabrata; the probe ofSEQ ID NO: 9 may be used in combination with a probe having the sequenceof any one of SEQ ID NOs: 20 to 25 for detecting Candida parapsilosis;and the probe of SEQ ID NO: 10 may be used in combination with a probehaving the sequence of any one of SEQ ID NOs: 15 to 17 for detectingCandida krusei.

The NMR based detection methods may also include an optionalamplification step, which may be performed to amplify the target nucleicacid prior to or substantially during contacting of the biologicalsample with the probe-nanoparticle conjugate. The amplification step ofthis method may include one or more of the following steps:

-   -   (a) performing one or more cycles of amplification with one or        more of the Candida specific PCR primers described above in a        detection tube,    -   (b) addition of the probe-nanoparticle conjugate,    -   (c) exposing the amplification reaction mixture, or an aliquot        thereof, to conditions permitting the aggregation or        disaggregation of the superparamagnetic particles,    -   (d) detecting hybridization between probes and target nucleic        acid by NMR and measuring the NMR signal from the detection        tube,    -   (e) repeating steps (a)-(d) until a desired amount of        amplification is obtained; and    -   (f) on the basis of the result of step (d), quantifying the        amplicons present at the corresponding cycle of amplification.

The amplification can be performed using combinations of the pan-Candidauniversal primers. Optionally, probes having sequences of any one ormore of SEQ ID NOs: 6 to 10 and 13 to 31 may also be used as forwardand/or reverse primers (in a symmetric or an asymmetric amplificationreaction) or in combination with one of the pan-Candida primers (in asymmetric or an asymmetric amplification reaction) to produce a Candidaamplicon. The amplification may be performed using pairwise combinationsof one of the universal primers and a primer selected from any one ofSEQ ID NOs: 6 to 10 and 13 to 31 (or its complement). Such pairwisecombinations may be designed based on the desired Candida speciesamplicon and the genomic positions of the probe and the primer sequencesto be used. Such pairwise combinations may include, without limitation,use of the universal forward primer in combination with: a) a reverseprimer having the complement of any one of SEQ ID NOs: 6 or 13 foramplifying a Candida albicans amplicon; b) a reverse primer having thecomplement sequence any one of SEQ ID NOs: 7, 9, or 20 to 25 foramplifying a Candida tropicalis or Candida parapsilosis amplicon; c) areverse primer having the complement sequence of any one of SEQ ID NOs:8, 18, or 19 for amplifying a Candida glabrata amplicon; d) a reverseprimer having the complement sequence of any one of SEQ ID NOs: 10 or 15to 17 for amplifying a Candida krusei amplicon; e) a reverse primerhaving the complement sequence of any one of SEQ ID NO: 26 or 27 foramplifying a Candida dubliniensis amplicon; f) a reverse primer havingthe complement sequence of any one of SEQ ID NO: 28 or 29 for amplifyinga Candida lusitaniae amplicon; g) a reverse primer having the complementsequence of any one of SEQ ID NO: 30 or 31 for amplifying a Candidaguillermondi amplicon.

Alternatively, the universal reverse primer may be used in combinationwith: a) a forward primer having the sequence of any one of SEQ ID NO: 6or 13 for amplifying a Candida albicans amplicon; b) a forward primerhaving the sequence of any one of SEQ ID NOs: 7, 9, or 20 to 25 foramplifying a Candida tropicalis or Candida parapsilosis amplicon; c) aforward primer having the sequence of any one of SEQ ID NOs: 8, 18, or19 for amplifying a Candida glabrata amplicon; d) a forward primerhaving the sequence of any one of SEQ ID NOs: 10 or 15 to 17 foramplifying a Candida krusei amplicon; e) a forward primer having thesequence of any one of SEQ ID NOs: 26 or 27 for amplifying a Candidadubliniensis amplicon; f) a forward primer having the sequence of SEQ IDNO: 28 or 29 for amplifying a Candida lusitaniae amplicon; g) a forwardprimer having the sequence of any one of SEQ ID NO: 30 or 31 foramplifying a Candida guillermondi amplicon.

The probe-nanoparticle conjugate can be added to the amplification mixprior to, or during, the PCR reaction, such that the probe-nanoparticleconjugates are present during the PCR reaction and under appropriateconditions can bind with the target nucleic acid. Alternatively, theprobe-nanoparticle conjugate can be added to the Candida lysate afterthe amplification step to detect the amplified Candida target nucleicacid.

The term “magnetic particle” refers to particles including materials ofhigh positive magnetic susceptibility such as paramagnetic compounds,superparamagnetic compounds, and magnetite, gamma ferric oxide, ormetallic iron. The magnetic nanoparticles may have a mean diameter offrom 150 nm to 1200 nm (for e.g., 150, 250, 350, 450, 550, 650, 750,850, 950, 1050, 1150, and 1200 nm) and are typically a compositematerial including multiple metal oxide crystals and an organic matrix.The magnetic particles of the invention may have a surface decoratedwith functional groups that can be conjugated to, e.g., one or morenucleic acid probes of having the sequence of any one of SEQ ID NOs: 6to 10 and 13-31, their complements, or probes having at least 90%, 95%,97%, 99%, or more sequence identity to the sequences of SEQ ID NOs: 6 to10 and 13-31 and their complements. The base particle for use in themethods of the invention can be any of the commercially availableparticles further described in, e.g., Table 2 of the InternationalApplication No. PC/US2011/56933. The magnetic nanoparticle maybemodified using protein blockers, as described in the InternationalApplication No. PCT/US2011/56933, in order to reduce non-specificbinding.

The nucleic acid probes of the invention can be linked to the metaloxide of the nanoparticle through covalent attachment to afunctionalized polymer or to non-polymeric surface-functionalized metaloxides. In the latter method, the magnetic particles can be synthesizedaccording to the method of, e.g., Albrecht et al., (Biochimie, 80:379,1998; incorporated herein by reference), which includes couplingdimercapto-succinic acid to the iron oxide to provide a carboxylfunctional group.

Where the probes of the invention are attached to magnetic particles viaa functionalized polymer associated with the metal oxide, preferably thepolymer is hydrophilic. In certain embodiments, the probe-conjugatednanoparticles may be made-using probes that have terminal amino,sulfhydryl, or phosphate groups and superparamagnetic iron oxidemagnetic particles bearing amino or carboxy groups on a hydrophilicpolymer. Several methods for synthesizing carboxy and aminoderivatized-magnetic particles are known in the art.

Probes of the invention may also be attached to a magnetic nanoparticlevia ligand-protein binding interaction, such as biotin-streptavidin, inwhich the ligand is covalently attached to the oligonucleotide and theprotein to the particle, or vice versa. This approach can allow for morerapid reagent preparation for NMR assays.

Amplification of Nucleic Acid Molecules of Candida

As is discussed above, the methods of the invention can optionallyinclude an amplification step in which specific regions of the Candidagenome are amplified to form a Candida species amplicon.

The terms “amplification” or “amplify” or derivatives thereof as usedherein to mean one or more methods known in the art for copying a targetor template nucleic acid, thereby increasing the number of copies of thetarget or template nucleic acid. Amplification may be exponential orlinear. Amplification may also be asymmetric (only the sense orantisense strand is copied) or symmetric (both the sense and antisensestrands are copied). Nucleic acid molecules amplified in this mannerform an “amplified region” or “amplicon.”

The amplification reaction increases the number of copies of Candidatarget nucleic acid molecules present in a Candida lysate solution.Preferably, amplification of the Candida target nucleic acid moleculesproduces a lysate solution that includes from 40% (w/w) to 95% (w/w)target nucleic acid (e.g., from 40 to 60%, from 60 to 80%, or from 85 to95% (w/w) target nucleic acid) and from 5% (w/w) to 60% (w/w) nontargetnucleic acid (e.g., from 5 to 20%, from 20 to 40%, or from 40 to 60%(w/w) nontarget nucleic acid). The species-specific nucleic acid probesdescribed above can be added to the lysate solution (prior to or afteramplification) for hybridization to the target nucleic acid moleculesand for detection of specific Candida species present in the biologicalsample.

In the context of the present invention, amplification produces Candidaspecies amplicons. For example, as shown in FIG. 6, amplification withthe pan-Candida primers produces an amplicon which has regions whichdiffer in sequence between Candida species and regions which share acommon sequence between all Candida species. The regions that aredifferent can be used to differentiate between Candida species.

Primers for amplification can be readily designed by those skilled inthe art to target a specific template nucleic acid sequence. In certainpreferred embodiments, the resulting amplicons are short and allow forrapid cycling and generation of copies. The size of the amplicon canvary as needed to provide the ability to discriminate target nucleicacids from non-target nucleic acids. For example, amplicons can be lessthan about 1,000 nucleotides in length. Desirably the amplicons are from100 to 500 nucleotides in length (e.g., 100 to 200, 150 to 250, 300 to400, 350 to 450, or 400 to 500 nucleotides in length).

While the exemplary methods described hereinafter relate toamplification using polymerase chain reaction (“PCR”), numerous othermethods are known in the art for amplification of nucleic acids (e.g.,isothermal methods, rolling circle methods). Alternate methods foramplification are described in further detail in, e.g., theInternational Application No. PCT/US2011/56933 and InternationalApplication No. PCT/US2011/56936.

Amplification may be performed by methods including a polymerase chainreaction (PCR) and the amplification reaction mixture may include, e.g.,(1) the target nucleic acid molecule(s), (2) forward and/or reverseamplification primers specific for the target nucleic acid molecule(s),and (3) a detection probe(s) that can bind to target sequences withinthe amplified target nucleic acid (the amplicon). In certainembodiments, the invention features the use of polymerase enzymescompatible with complex biological samples such as whole blood, e.g.,NEB Hemoklentaq, DNAP Omniklentaq, Kapa Biosystems whole blood enzyme,Thermo-Fisher Finnzymes Phusion enzyme.

In certain embodiments, the primers can include the pan-Candida primersequences where the forward and reverse primer have the sequence 5′-GGCATG CCT GTT TGA GCG TC-3′ (SEQ ID NO: 11) and the sequence 5′-GCT TATTGA TAT GCT TAA GTT CAG CGG GT-3′ (SEQ ID NO: 12), respectively. Inother embodiments, specific Candida species amplicons may be amplifiedby the use of appropriate forward and reverse primers that bind to andproduce species-specific Candida amplicons, as described above.

In certain embodiments the amplification may be asymmetric, in which theconcentration of one of the primers in the reaction is higher than theconcentration of the other primer. For example, the concentration of theforward amplification primer may be about 4 times (e.g., 2, 3, 4, 5, 10,20, 50, times) the concentration of the reverse primer, or vice-versa.This reaction condition results in preferential amplification of singlestranded amplicons. In other embodiments, the amplification may besymmetric. In this case, the PCR reaction may be performed using equalconcentrations of the forward and reverse primers, which results indouble stranded amplicons. Methods for performing asymmetric andsymmetric PCR are known in the art (see, for example, Poddar, Molecularand Cellular Probes 14: 25-32, 2000, incorporated herein by reference).In one particular example, the pan-Candida universal forward and/orreverse primers can be used in pairwise combinations with any one of theprimers having the sequence of SEQ ID NOs: 6-10 and 13-31, or theircomplements, to produce species-specific Candida amplicons.

Biological Samples that can be Tested for the Presence of a CandidaSpecies

The compositions, methods, and kits featured in the invention may beused to detect at least one (and preferably two or more) Candida speciesthat may be present in a biological sample. By biological sample ismeant a variety of media including, but not limited to, biofluids,tissue samples, culture samples (e.g., a blood culture), food products,water samples, and soil samples that contain one or more species ofCandida cells. For example, the biological sample can be a sample whichhas been processed to remove cellular and tissue debris and patientnucleic acids, or the biological sample may be a sample that has beenprocessed to partially or fully isolate Candida cells from the sample.The biological sample may include, e.g., cell suspensions or celllysates. The biological sample may also be one that has beensubstantially unprocessed, such as a whole blood sample. The biologicalsample may also be any sample in which the nucleic acid molecules ofCandida have been substantially isolated, enriched by amplification orother methods, or purified. The biological sample can be any sample thatcontains at least one genome equivalent of Candida.

Typically, the sample is human in origin, but alternatively it maybenon-human in origin (such as from an animal). The biological sample ispreferably a fluid sample, and can typically comprise a body fluid, butmay also be a solid tissue sample. The sample may be, e.g., whole blood,sweat, tears, urine, saliva, semen, serum, plasma, cerebrospinal fluid(CSF), feces, vaginal fluid, sputum, nasopharyngeal aspirate or swab,and lacrimal fluid. The sample can also be mucous or epithelial swab(buccal swab), tissues, organs, bone, teeth, and tumors.

The compositions, methods, and kits of the invention can also be used tomonitor and diagnose infectious disease in a multiplexed, automated, nosample preparation system. Examples of pathogens that may be detectedusing the devices, kits, and methods of the invention include, e.g.,Candida, e.g., C. albicans, C. glabrata, C. krusei, C. tropicalis, C.parapsilosis, C. dubliniensis, C. lusitaniae, and C. guillermondi. Themethods of the invention can be used to identify and monitor thepathogenesis of disease in a subject, to select therapeuticinterventions, and to monitor the effectiveness of the selectedtreatment.

The methods of the invention an also be used to monitor sepsis or septicshock. Sepsis and septic shock are serious medical conditions that arecharacterized by a whole-body inflammatory state (systemic inflammatoryresponse syndrome or SIRS) and the presence of a known or suspectedinfection. Sepsis is defined as SIRS in the presence of an infection,septic shock is defined as sepsis with refractory arterial hypotensionor hypoperfusion abnormalities in spite of adequate fluid resuscitation,and severe sepsis is defined as sepsis with organ dysfunction,hypoperfusion, or hypotension. To determine whether a patient hassepsis, it is necessary to identify the presence of a pathogen and toidentify bacterial or fundal origin. To most effectively treat apatient, the earliest initiation of appropriate therapy is important toa satisfactory outcome. Antimicrobial and other treatments for sepsisrely on the classification of pathogens at multiple levels, includingthe identification of an agent as 1) bacterial, viral, fungal, parasiticor otherwise; 2) gram positive, gram negative, yeast, or mold, 3)species, and 4) susceptibility.

Each of these levels of specificity improves the time to initiation ofappropriate therapy, and each step further down the track will lead to anarrowing of therapeutic agents to the most specific set. Withoutabsolute susceptibility data; empiric approaches to care rely on theinformation available about the pathogen (at whichever level) and thepattern of pathogen frequency and susceptibility trends in the hospitalof another site of care. Thus, certain categories of pathogens arefrequently presumed to be causative until there are more data to refinethe pairing of pathogen and therapy. The methods of the presentinvention can be used to facilitate early diagnosis of fungal (e.g.,Candida) infection, and thus can provide improved therapeutic outcomesin patients diagnosed using such methods.

Biological Sample Lysis Methods

In any of the methods of the invention for detection of Candida speciesin a biological sample, the disruption of patient cells present in thebiological sample can be carried out using a lysis agent (e.g., a lysisbuffer, a hypotonic buffer, or a nonionic detergent). Lysis bufferswhich can be used in the methods of the invention include, withoutlimitation, isotonic solutions of ammonium chloride (optionallyincluding carbonate buffer and/or EDTA), and hypotonic solutions.Alternatively, the lysis agent can be aqueous solution of one or moredetergents (e.g., nonionic, ionic, polymeric, and zwitterionic, such as,(e.g., nonyl phenoxypolyethoxylethanol (NP-40), 4-octylphenolpolyethoxylate (Triton-X100), Brij-58), or related surfactants, andmixtures thereof). The lysis agent disrupts at least some of the samplecells, allowing a large fraction of certain components to be separated.For example, if the biological sample is whole blood, the lysis agentdisrupts at least some of the red blood cells present in the sample,allowing a large fraction of certain components of whole blood (e.g.,certain whole blood proteins) to be separated (e.g., as supernatantfollowing centrifugation) from the Candida fungal cells present in thewhole blood sample. The Candida fungal cells can then be optionallywashed to further remove whole blood lysed debris from the sample. Thelysed sample may then be centrifuged to produce a supernatant and apellet. The resulting pellet containing the Candida cells may bereconstituted to form a suspension of Candida cells, which may befurther treated as discussed below.

Fungal Lysis Methods

In any of the methods of the invention for detection of Candida speciesin a biological sample, the isolated Candida cells in suspension may belysed using either chemical or mechanical methods known in the art inorder to release the target nucleic acid from the Candida cells. Forexample, chemical lysis methods include, without limitation, treatmentof fungal cells with one or more enzymes, such as Zymolyase (see, e.g.,Park et al., J. Clin Microbiol. 38: 2829-2839, 2000, incorporated hereinby reference), or detergents, or surfactants. Alternatively, Candidacells may be lysed using mechanical methods known in the art. Forexample, mechanical lysis methods include, without limitation, use ofsolid particles, sand, or glass shards, use of glass beads as describedin, e.g., Hirose et al., Biotechnology Techniques 13: 571-575, 1999(incorporated herein by reference), use of beads with sonication ormechanical vortex centrifugation or magnetic vortex centrifugation asdescribed in, e.g., U.S. Pat. No. 7,723,095 (incorporated herein byreference), bead beating described in International Application No.PCT/US2011/56933 and International Application No. PCT/US2011/56936, useof a finned tube, e.g., use of a finned tube with beads added to thetube for lysing the sample, as described in U.S. Patent Application No.61/601,842 (incorporated herein by reference), use of ultrasound asdescribed in, e.g., U.S. Pat. No. 6,686,195 (incorporated herein byreference), heating the sample or applying high pressure as describedin, e.g., Chisti and Moo-Young, Biotechnology/The Science and Business,Chapter 13, Harwood Academic Publishers, 1999 (incorporated herein byreference), use of solid particles in the presence of chelating agentsas described in, e.g., U.S. Pat. No. 7,494,771 B2 (incorporated hereinby reference), use of high speed agitation bead mills as described in,e.g., Kula and Schutte, Biotechnology Progress 3(1): 31, 1987(incorporated herein by reference), use of cell wall breaking devices asdescribed in, e.g., U.S. Pat. No. 4,295,613 (incorporated herein byreference), and freeze-boil and freeze-thaw methods (see, e.g.,Griffiths et al., J. Med Microbiol. 55: 1187-1191, 2006, incorporatedherein by reference).

When beads are used to lyse the Candida cells, the beads may be 0.5 mmglass beads, 0.7 mm silica beads, 0.1 mm silica beads, 0.7 mm silicabeads, yttrium stabilized zirconium oxidized beads, or a mixture ofdifferently sized beads made of inert material (see, e.g., Curran andEvans, J. Bacteriol. 43(2): 125, 1942; and Lamanna and Mallette, J.Bacteriol. 67(4): 503, 1954, incorporated herein by reference).Typically, the beads are added to the tube containing the Candida cellsin suspension and the tube is placed in a vortexer (e.g., a Biospec beadbeater) and vortexed at maximum speed for 5-15 minutes (e.g., 5 minutes)so that the Candida cells are lysed and the endogenous target nucleicacids are released.

If desired, the lysate can be further processed to purify the Candidanucleic acid molecules by methods known in the art which include,without limitations, use of an ion exchange columns, e.g., an anionexchange column, such as a Qiagen column, phenol-chloroform extractionand ethanol precipitation methods as described in Maniatis et al.“Molecular Cloning—A laboratory manual” Cold Spring Harbor Press, 1982(incorporated herein by reference), use of fungal DNA extraction kits asdescribed in, e.g., Fredricks et al., J. Clin. Microbiol. 43 (10):5122-5128, 2005 (incorporated herein by reference), use of the “CTABmethod” as described in Zhang et al., FEMS Microbiology Letters 145(2):261-265, 1996 (incorporated herein by reference), and spry technology.

Kits for Detection of Candida Species

Kits of the invention may include one or more probes and primers havingthe sequence of any of SEQ ID NOs: 1 to 41 (e.g., SEQ ID NOs: 1 to 5)for detecting Candida species in biological samples. The kits mayfurther include instructions for using one or more of the probes andprimers in methods for detecting a Candida species (e.g., Candidaalbicans, Candida tropicalis, Candida krusei, Candida glabrata, Candidaparapsilosis, Candida dubliniensis, Candida lusitaniae, and Candidaguillermondi) in a biological sample.

In addition, the kits may include any combination of reagents for samplelysis, fungal cell lysis, amplification of Candida nucleic acidmolecules, and reagents for detection of Candida species appropriate forfluorescence detection and/or NMR based detection.

The kits may also include one or more reagents for fluorescentlylabeling a probe or probes (e.g., probes having SEQ ID NOs: 1 to 5) orthe kits may include a probe or probes that are already fluorescentlylabeled (e.g., the probes of the kits may be molecular beacon probeshaving the sequence of any one of SEQ ID NOs; 1 to 5). The kits may alsoinclude reagents for conjugating nanoparticles to a probe or probes ofthe invention (e.g., a probe having the sequence of any one of SEQ IDNOs: 6 to 10 and 13 to 31) or the kits may include a probe or probesthat are already conjugated to nanoparticles for use in NMR baseddetection assays.

Uses for Candida Specific Molecular Beacon Probes

The Candida specific molecular beacon probes described herein can beused in several ways, including the following:

-   -   a) If intact fungal cells can be enriched and/or partially or        fully purified from a biological matrix (e.g., a biological        sample, such as whole blood, serum, sputum, urine, etc.), the        Candida specific molecular beacon probes can be used in a method        to detect and identify a fungal species in the biological        matrix;    -   b) If fungal DNA can be isolated from any biological sample, the        Candida specific molecular beacon probes can be used to detect        the Candida DNA, as well as identify the particular fungal        species;    -   c) In a rapid and convenient method for fungal identification in        a post blood-culture sample. This could be through direct        detection, using the Candida specific molecular beacon probes,        of Candida within a small aliquot (˜5-50 uL) of the blood        culture media and/or removal of 5 mL or less of the media,        centrifugation and removal of the supernatant, and resuspension        of the cells within TE. This potentially could be done at        earlier time points during blood culture incubation to maximize        time to result;    -   d) In a robust and quantitative assay for quantification of        fungal cells used to generate Candida spiked whole blood samples        from frozen cell bullets;    -   e) In a high throughput and sensitive tool to quantify and        ensure lot to lot reproducibility and stability of Candida        external controls; and    -   f) In a high throughput and sensitive orthogonal assay to        monitor the enzymatic activity of a polymerase for use in an        amplification reaction using Candida specific nucleic acid        molecules and/or to monitor the activity and reliability of a        PCR master mix (lot to lot reproducibility, shelf life        stability, material compatibility, and ship stability).

The following examples are meant to illustrate the invention. They arenot meant to limit the invention in any way.

EXAMPLES Example 1: Detection of Candida krusei Using a Species-SpecificMolecular Beacon Probe in a Real-Time PCR Assay

A general protocol for detecting Candida species using fluorescentlylabeled molecular beacon probes (e.g., SEQ ID NOs: 1 to 5) is describedbelow. For preparation of Candida cell lysate, 1 mL of PBS, TE, or YPDbroth containing spiked Candida cells at concentrations ranging from 10⁴to 1 Candida cells were added to a tube containing 300 mg of yttriumstabilized zirconium oxide beads. The cells were harvested viacentrifugation at 6000G for 5 minutes and the supernatant was removed.The cells were then washed with 150 to 1500 μL of TE buffer andharvested again via centrifugation at 60000. 100 μL of TE buffer wasadded to the tubes and the tube was placed in a vortexer (Biospec beadbeater) at maximum speed for 5 minutes. The resulting Candida lysate wasremoved and transferred to a clean 1.7 mL polypropylene tube, and waseither used directly in a real-time PCR reaction or was furtherprocessed using a Qiagen kit to isolate the Candida genomic DNAcontaining the target nucleic acid molecules.

An asymmetric master mix was prepared using pan-Candida forward andreverse primers, as shown in Table 1.

TABLE 1 Component per 100 uL reaction Nuclease free water* 17.33 5Xreaction Buffer 20 10 mM mix 2 100 uM Forward Primer 0.3 100 uM ReversePrimer 0.075 100 uM Molecular beacon 0.3 Hemoklentaq 10 sum 50.00

The molecular beacon probe (at a concentration of 300 nM; the molecularbeacon probe may be used at a concentration of ˜100-600 nM) was addeddirectly to the asymmetric master mix. Fifty microliters of cell lysateor diluted genomic DNA was added directly to the master mix in a 96-wellplate and the plate was loaded onto a real-time PCR thermal cycler (i.e.Roche LightCycler). The real-time cycling parameters used for asymmetricamplification are described in Table 2.

TABLE 2 Pre-incubation: 95 C., 5 minutes, ramp rate: 4.4 degrees C./secAmplification-45 cycles Denaturation: 95 C., 20 sec, ramp rate: 4.4degrees C./sec Annealing: 60 C., 40 sec, ramp rate: 2.2 degrees C./secElongation: 68 C., 30 sec, ramp rate: 4.4. degrees C./sec Melt Curve 95C., 1 minute, ramp rate: 4.4 degrees C./sec 40 C., 1 minute, ramp rate:2.2 degrees C./sec 80 C., continuous, ramp rate: 0.29 degrees C./sec

In this example, a molecular beacon probe specific for Candida krusei(having the sequence of SEQ ID NO: 5) was used. Real-time PCR data areprovided below for reactions containing 0 to 50,000 genomic equivalentsof isolated Candida krusei genomic DNA added as template to eachreaction. Four replicates for each reaction condition were performed andthe mean Cp values (cross-over point), standard deviation of Cp (Std.Cp) and CV (coefficient of variation, n=3 or n=4) were calculated foreach experiment described below. The average amplification efficiencyand the PCR efficiency for the reactions were also calculated in eachcase to monitor that the results were not affected by amplificationartifacts.

As shown in Table 3, and FIG. 2A, the Candida krusei molecular beaconprobe was used successfully to detect the target nucleic acid moleculein the genomic DNA isolated from Candida krusei. The “ND” indicates “notdetected” and no Cp was reported.

TABLE 3 C. krusei genomic equivalents/rx Cp1 Cp2 Cp3 Cp4 Average Std CpCV 50,000 21.36 21.29 21.34 21.5 21.37 0.09 0.4% 5,000 24.91 24.86 24.9424.87 24.90 0.04 0.1% 500 28.23 28.27 28.11 28.33 28.24 0.09 0.3% 5031.81 31.74 31.78 31.59 31.73 0.10 0.3% 5 35.09 34.98 34.95 35.19 35.010.11 0.3% no template ND ND ND ND ND amplification  1.96 efficiency =PCR efficiency = 96%

The Candida krusei specific molecular beacon probe was also used todetect C. krusei in a Candida lysate prepared as described above.Real-time PCR data are provided below for PCR reactions containingCandida lysate prepared using different concentrations of Candida cells.The average amplification efficiency and the PCR efficiency for thereactions were also calculated to determine whether use of a lysate asthe source of the genetic material affected the amplification and PCRefficiency.

As seen in Table 4 below, the amplification and PCR efficiencies whenlysate was used are similar to the results obtained when isolatedgenomic DNA template was used (see Table 3). Thus, genetic material froma Candida cell lysate can be directly used in a PCR reaction to amplifyCandida target nucleic acid molecules. Also, as shown in FIG. 2B,Candida krusei was successfully detected in a sample prepared as aCandida lysate using the C. krusei specific molecular beacon probe.

TABLE 4 C. krusei 1rst run 2nd run Std cells/rx Cp1 Cp2 Cp3 Cp4 Cp1 Cp2Cp3 Average Cp CV 400 27.98 27.96 27.88 27.9 28.01 28.03 27.96 27.960.05 0.2% 40 31.26 31.25 31.46 31.24 31.53 31.51 31.51 31.39 0.14 0.4% 434.34 34.13 33.96 34.5 34.34 34.86 34.59 34.39 0.30 0.9% amplification 2.05 efficiency = PCR efficiency = 105%

Additional data showing the successful use of a HEX-labeled C. kruseiprobe with genomic Candida krusei DNA are shown in Table 5 and FIG. 3C,and additional data showing the successful use of a HEX-labeled C.krusei probe with Candida krusei are shown in Table 6 and FIG. 3F. Thevolume of diluted genomic DNA or lysate added in these PCR reactions was100 ul instead of 50 ul as in the reactions described above.

TABLE 5 C. krusei gDNA titration_300 nM HEX copies/rx Cp1 Cp2 Cp3 Cp4Average Std Cp CV 10,000 24.77 24.84 24.8 24.8 24.79 0.04 0.1% 1,00028.34 28.29 28.3 28.4 28.32 0.04 0.1% 100 31.79 31.8 31.8 31.9 31.810.03 0.1% 10 35.07 35.01 35.2 35.1 35.08 0.07 0.2% no template ND ND NDamplification  1.95 efficiency PCR efficiency 95%

TABLE 6 C. krusei cell lysate_300 nM HEX genomic equivalent/rx Log Cp1Cp2 Cp3 Cp4 Average Std Cp CV 1,000 3.0 27.81 27.59 27.9 27.8 27.76 0.120.4% 100 2.0 31.21 31.1 31.2 31.1 31.15 0.05 0.2% 10 1.0 34.72 34.8834.9 34.9 34.86 0.10 0.3% no cell lysate 17.9 12.3 15.05 3.96 26.3%amplification efficiency  1.91 PCR efficiency 91%

Example 2: Detection of C. parapsilosis Using a Species-SpecificMolecular Beacon Probe in a Real-Time PCR Assay

A HEX labeled C. parapsilosis probe (having the sequence of SEQ ID NO:4) was used at 300 nM concentration in a real-time PCR assay that wasperformed according to the parameters described in Table 1 above. 100 μlof diluted genomic DNA was added to the PCR reaction mix and 45 cyclesof amplification were performed with an annealing temperature of 55° C.A titration using different amounts of genomic DNA template wasperformed. Real-time PCR data are provided in Table 7 below forreactions containing 0 to 10,000 copies of genomic DNA per reaction.Four replicates for each reaction condition were performed and theaverage Ct values were calculated. The average amplification efficiencyand the PCR efficiency for the reactions were also calculated. Table 7and FIG. 3A show that the C. parapsilosis molecular beacon probe wasused successfully to detect the target nucleic acid molecule usinggenomic DNA isolated from Candida parapsilosis.

TABLE 7 C. parapsilosis gDNA titration_300 nM HEX copies/rx Log Cp1 Cp2Cp3 Cp4 Average Std Cp CV 10,000 4.0 25.01 24.96 24.99 25.09 25.01 0.060.2% 1,000 3.0 28.45 28.5 28.44 28.48 28.47 0.03 0.1% 100 2.0 31.8431.81 31.85 31.83 31.83 0.02 0.1% 10 1.0 35.27 35.06 34.99 34.74 35.020.22 0.6% no ND ND ND template amplification  1.99 efficiency PCRefficiency 99%

Next, a similar assay was performed using a Candida parapsilosis lysaterather than genomic DNA. A titration was performed with 100 ul of aCandida lysate prepared with different concentrations of C. parapsilosiscells. Real-time PCR data are provided below (Table 8) for PCR reactionscontaining Candida lysate having 0 to 1000 (i.e., 0, 10, 100, or 1,000)genomic equivalents of Candida parapsilosis genomic DNA per reaction. Asshown in Table 8 and FIG. 3D, Candida parapsilosis was successfullydetected using the C. parapsilosis specific molecular beacon probe atall genomic equivalents prepared using a Candida lysate.

TABLE 8 C. parapsilosis cell lysate_300 nM HEX genomic Std equivalent/rxLog Cp1 Cp2 Cp3 Cp4 Average Cp CV 1,000 3.0 28.61 28.65 28.73 28.6628.66 0.05 0.2% 100 2.0 31.88 31.89 31.97 31.93 31.92 0.04 0.1% 10 1.034.91 34.96 34.9 34.97 34.94 0.04 0.1% no cell lysate amplification 2.08 efficiency PCR 108% efficiency

Example 3: Detection of C. tropicalis Using a Species-Specific MolecularBeacon Probe in a Real-Time PCR Assay

A HEX labeled C. tropicalis probe (having the sequence of SEQ ID NO: 2)was used at 300 nM concentration in a real-time PCR assay that wasperformed according to the parameters described in Table 1 above. 100 μlof diluted genomic DNA was added to the PCR reaction mix and 45 cyclesof amplification were performed with an annealing temperature of 55° C.A titration using different amounts of genomic DNA template wasperformed. Real-time PCR data are provided in Table 9 for reactionscontaining 0 to 10,000 (i.e., 0, 100, 1,000, and 10,000) copies ofgenomic DNA per reaction. Four replicates for each reaction conditionwere performed and the average Ct values were calculated. The averageamplification efficiency and the PCR efficiency for the reactions werealso calculated. Table 9 and FIG. 3B show that the C. tropicalismolecular beacon probe was used successfully to detect the targetnucleic acid molecule using genomic DNA isolated from Candidatropicalis.

TABLE 9 C. tropicalis gDNA titration_300 nM HEX copies/rx Cp1 Cp2 Cp3Cp4 Average Std Cp CV 10,000 25.41 25.03 24.99 25.26 25.17 0.20 0.8%1,000 28.79 28.72 28.82 28.69 28.76 0.06 0.2% 100 32.21 32.23 32.2532.32 32.25 0.05 0.1% 10 35.11 35 34.96 35.38 35.11 0.19 0.5% notemplate ND ND ND amplification  2.00 efficiency PCR efficiency 100%

Next, a similar assay was performed using a Candida tropicalis lysaterather than genomic DNA. A titration was performed with 100 ul of aCandida lysate prepared with different concentrations of C. tropicaliscells. Real-time PCR data are provided below (Table 10) for PCRreactions containing Candida lysate having 0 to 1000 (i.e., 0, 10, 100,or 1,000) genomic equivalents of Candida tropicalis genomic DNA perreaction. As shown in Table 10 and FIG. 3E, Candida tropicalis wassuccessfully detected using the C. tropicalis specific molecular beaconprobe at all genomic equivalents prepared using a Candida lysate.

TABLE 10 C. tropicalis cell lysate_300 nM HEX genomic Std equivalent/rxLog Cp1 Cp2 Cp3 Cp4 Average Cp CV 1,000 3.0 28.57 28.67 28.59 28.6128.61 0.04 0.2% 100 2.0 31.99 31.92 32.06 32.05 32.01 0.06 0.2% 10 1.035.07 34.57 34.55 34.46 34.66 0.28 0.8% no cell lysate ND ND NDamplification  2.14 efficiency PCR 114% efficiency

Example 4: Detection of C. albicans Using a Species-Specific MolecularBeacon Probe in a Real-Time PCR Assay

A HEX labeled C. albicans probe (having the sequence of SEQ ID NO: 1)was used at 300 nM concentration in a real-time PCR assay that wasperformed according to the parameters described in Table 1 above. 100 μlof diluted genomic DNA was added to the PCR reaction mix and 45 cyclesof amplification were performed with an annealing temperature of 60° C.A titration using different amounts of genomic DNA template wasperformed. Real-time PCR data are provided in Table 11 for reactionscontaining 0 to 10,000 copies (i.e., 0, 10, 100, 1,000, or 10,000) ofgenomic DNA per reaction. Four replicates for each reaction conditionwere performed and the average Ct values were calculated. The averageamplification efficiency and the PCR efficiency for the reactions werealso calculated. Table 11 and FIG. 4A show that the C. albicansmolecular beacon probe was used successfully to detect the targetnucleic acid molecule using genomic DNA isolated from Candida albicans.

TABLE 11 C. albicans genomic DNA Std copies/rx Log Cp1 Cp2 Cp3 Cp4Average Cp CV 10,000 4.0 24.57 24.71 24.7 25 24.75 0.18 0.7% 1,000 3.028.01 28.15 28.19 28.45 28.20 0.18 0.7% 100 2.0 31.59 31.63 31.65 31.8631.68 0.12 0.4% 10 1.0 34.86 34.14 35.02 35.75 34.94 0.66 1.9% notemplate ND ND ND amplification  1.97 efficiency PCR 97% efficiency

Next, a similar assay was performed using a Candida albicans lysaterather than genomic DNA. A titration was performed with 100 ul of aCandida lysate prepared with different concentrations of C. albicanscells. Real-time PCR data are provided below (Table 12) for PCRreactions containing Candida lysate which provide 0 to 400 (i.e., 0, 4,40, or 400) genomic equivalents of Candida albicans genomic DNA perreaction. As shown in Table 12 and FIG. 4C, Candida albicans wassuccessfully detected using the C. albicans specific molecular beaconprobe at all genomic equivalents prepared using a Candida lysate.

TABLE 12 C. albicans cell lysate Std cells/rx Log Cp1 Cp2 Cp3 Cp4Average Cp CV 400 2.6 28.59 28.68 28.66 28.68 28.65 0.04 0.1% 40 1.632.15 32.1 32.02 32.02 32.07 0.06 0.2% 4 0.6 34.84 34.86 34.66 34.8634.81 0.10 0.3% no cell lysate 16.27 16.27 ND ND amplification  2.11efficiency PCR 111% efficiency

Example 5: Detection of C. glabrata Using a Species-Specific MolecularBeacon Probe in a Real-Time PCR Assay

A HEX labeled C. glabrata probe (having the sequence of SEQ ID NO: 3)was used at 300 nM concentration in a real-time PCR-assay that wasperformed according to the parameters described in Table 1 above. 100 μlof diluted genomic DNA was added to the PCR reaction mix and 45 cyclesof amplification were performed with an annealing temperature of 60° C.A titration using different amounts of genomic DNA template wasperformed. Real-time PCR data are provided in Table 13 for reactionscontaining 0 to 10,000 (i.e., 0, 10, 100, 1,000, or 10,000) copies ofgenomic DNA per reaction. Four replicates for each reaction conditionwere performed and the average Ct values were calculated. The averageamplification efficiency and the PCR efficiency for the reactions werealso calculated. Table 13 and FIG. 4B show that the C. glabratamolecular beacon probe was used successfully to detect the targetnucleic acid molecule using genomic DNA isolated from Candida glabrata.

TABLE 13 C. glabrata genomic DNA Std copies/rx Log Cp1 Cp2 Cp3 Cp4Average Cp CV 10,000 4.0 26.55 26.38 26.38 26.46 26.44 0.08 0.3% 1,0003.0 30.02 30.05 30.02 30.18 30.07 0.08 0.3% 100 2.0 33.02 32.97 32.7832.84 32.90 0.11 0.3% 10 1.0 36.47 34.22 36.73 36.72 36.04 1.22 3.4% notemplate ND ND ND amplification  2.07 efficiency PCR 107% efficiency

Next, a similar assay was performed using a Candida glabrata lysaterather than genomic DNA. A titration was performed with 100 ul of aCandida lysate prepared with different concentrations of C. glabratacells. Real-time PCR data are provided below (Table 14) for PCRreactions containing Candida lysate which provide 0 to 400 (i.e., 0, 4,40, or 400) genomic equivalents of Candida glabrata genomic DNA perreaction. As shown in Table 14 and FIG. 4D, Candida glabrata wassuccessfully detected using the C. glabrata specific molecular beaconprobe at all genomic equivalents prepared using a Candida lysate.

TABLE 14 C. glabrata cell lysate Std cells/rx Log Cp1 Cp2 Cp3 Cp4Average Cp CV 400 2.6 29 29.15 29.24 28.95 29.09 0.13 0.5% 40 1.6 32.0732.11 31.92 32.02 32.03 0.08 0.3% 4 0.6 35.09 34.8 34.7 34.84 34.86 0.170.5% no cell lysate ND ND ND amplification 2.22 efficiency PCR 122%efficiency

The above data in Tables 4 to 14 and in FIGS. 3 and 4 demonstrate thateach of the species specific probes can be used to successfully detectspecific Candida species using either purified genomic DNA as an inputin a real-time PCR assay or, alternatively, using a fungal cell lysateas an input in the assay.

Example 6: Multiplexed Real-Time PCR Assay for Detecting MultipleCandida Species in a Sample

For a multiplexed molecular beacon assays, 300 nM of eachspecies-specific beacon probe (0.3 uL per 100 uL reaction) were added tothe PCR master mix. The C. parapsilosis and C. tropicalis molecularbeacons (SEQ ID NO: 4 and SEQ ID NO; 2, respectively) are labeled with aFAM and a HEX fluor, respectively. The beacons are multiplexed in thereaction and detected in their respective channels (488-533 nm for FAM)and (488-610 for HEX). To ensure that the amplification efficiencieswere similar between reactions containing a single beacon and reactionscontaining multiplexed beacons, C. tropicalis and C. parapsilosisgenomic DNA was titrated into reactions at concentrations spanning 1E4down to 10 copies per 20 uL reaction volume. These reactions includedeither a single beacon for detection (shown in Table 15 for C.parapsilosis and Table 17 for C. tropicalis) or the two beacons in thesame reaction but detected with different channels (Table 16 and Table18). In both cases the amplification effiencies were similar for boththe single detection reactions and multiplexed detection reactions withno measurable impact on detection sensitivity. Thus, both C. tropicalisand C. parapsilosis can be detected within a single PCR reaction andpotentially all five beacons could be multiplexed in a single reactionenabling detection of the five most clinically relevant Candida specieswithin the same well.

TABLE 15 Channel copies/ 483-533 C. parapsilosis detection single beaconrx Log Cp1 Cp2 Cp3 Average Std Cp CV 10000 4 21.58 21.53 21.5 21.536670.040415 0.2% 1000 3 25.01 24.96 24.97 24.98 0.026458 0.1% 100 2 28.5928.64 28.58 28.60333 0.032146 0.1% 10 1 31.89 31.81 32 31.9 0.0953940.3% no ND tem- plate amplification 1.9 efficiency = PCR efficiency =93.2%

TABLE 16 483- Channel 533 C. parapsilosis detection mulitplexed reactioncopies/rx Log Cp1 Cp2 Cp3 Average Std Cp CV 10000 4 22.61 22.51 22.4822.53333 0.068069 0.3% 1000 3 26.01 25.96 25.985 0.035355 0.1% 100 229.55 29.29 29.28 29.37333 0.15308 0.5% 10 1 32.62 32.67 32.98 32.756670.195021 0.6% no ND template amplification 1.96 efficiency = PCRefficiency = 96.0%

TABLE 17 488- Channel 610 C. tropicalis detection single beaconcopies/rx Log Cp1 Cp2 Cp3 Average Std Cp CV 10000 4 21.44 21.47 21.5921.5 0.079373 0.4% 1000 3 24.63 24.77 24.67 24.69 0.072111 0.3% 100 228.23 28.01 28.08 28.10667 0.112398 0.4% 10 1 31.92 31.65 31.86 31.810.141774 0.4% no ND template amplification 1.95 efficiency = PCRefficiency = 95.3%

TABLE 18 488- Channel 610 C. tropicalis detection multiplexed reactioncopies/rx Log Cp1 Cp2 Cp3 Average Std Cp CV 10000 4 21.55 21.57 21.5521.55667 0.011547 0.1% 1000 3 24.96 24.89 24.99 24.94667 0.051316 0.2%100 2 28.72 28.5 28.51 28.57667 0.124231 0.4% 10 1 32.12 31.84 31.8931.95 0.149332 0.5% no ND template amplification 1.94 efficiency = PCRefficiency = 94.0%

Example 7: DNA Sequencing-Based Detection of Candida Species

A probe having the sequence of SEQ ID NO: 32 (specific to C. krusei) wascontacted with nucleic acid molecules that were amplified from withinwhole blood lysate using the pan-Candida forward and reverse primers(SEQ ID NOs: 11 and 12), followed by conducting a phenol/chloroformextraction, a chloroform extraction, and then conducting Sanger DideoxySequencing using Big Dye Terminators on an AB 3730 capillary sequencinginstrument. The resulting chromatogram, which is shown in FIG. 7,demonstrates that this method can also be used to of detect Candidaspecies using the probes and primers of this invention.

The sequencing-based detection method described above can also beperformed using a probe having the sequence of SEQ ID NO: 33 to detectC. krusei; a probe having the sequence of SEQ ID NO: 34 or 35 to detectC. albicans; a probe having the sequence of SEQ ID NO: 36 or 37 todetect C. glabrata; a probe having the sequence of SEQ ID NO: 38 or 39to detect C. parapsilosis; or a probe having the sequence of SEQ ID NO:40 or 41 to detect C. tropicalis.

Example 8: Detection of C. albicans Using a Species-Specific MolecularBeacon Probe in a Real-Time PCR Assay

A FAM labeled C. albicans probe (having the sequence of SEQ ID NO: 1)was used at 300 nM concentration in a real-time PCR assay that wasperformed according to the parameters described in Table 1 above. 100 μlof diluted genomic DNA was added to the PCR reaction mix and 45 cyclesof amplification were performed with an annealing temperature of 60° C.A titration using different amounts of genomic DNA template wasperformed. Real-time PCR data are provided in Table 19 for reactionscontaining 0 to 50,000 copies (i.e., 0 (no template), 5, 50, 500, 5,000,or 50,000) of genomic DNA per reaction. Four replicates for eachreaction condition were performed and the average Cp values werecalculated. The average amplification efficiency (2.03) and the PCRefficiency (103%) for the reactions were also calculated. Table 19 showsthat the C. albicans molecular beacon probe was used successfully todetect the target nucleic acid molecule using genomic DNA isolated fromC. albicans.

TABLE 19 C. albicans detection in singleplex reactions Std copies/rx LogCp1 Cp2 Cp3 Cp4 Average Cp CV 50000 5 21.71 21.86 21.9 21.9 21.84 0.090.4% 5000 4 25.24 25.06 25.23 25.27 25.20 0.09 0.4% 500 3 28.66 28.6428.72 28.68 28.68 0.03 0.1% 50 2 31.84 31.87 31.88 31.84 31.86 0.02 0.1%5 1 34.39 35 35 35 34.80 0.31 0.9% no ND ND ND ND template slope −3.25660.31 amplification 2.03 efficiency = PCR efficiency = 103%

Next, a similar assay was performed using a C. albicans lysate ratherthan genomic DNA. To determine the ability of the molecular beacons todetect Candida within crude cell lysates, we prepared lysate fromCandida cells spiked in 1× phosphate buffered saline (PBS) buffer.Candida cells were quantified using a Coulter counter and were spikedinto PBS at concentrations ranging from ˜520 cells/mL to ˜3 cells/mL.The in-vitro spiked solutions were added to a 1 mL polypropylene tubecontaining 300 mg of yttrium stabilized zirconium oxide beads. The cellswere harvested via centrifugation at 6000×G for 5 minutes and thesupernatant was removed. 100 uL of 1×TE was added to the tubes and thetubes were subjected to bead beating, as described herein, by placementin a vortexer (Biospec bead beater) at maximum speed for 5 minutes.Fifty microliters of lysate was then transferred into an asymmetric PCRmaster mix containing the molecular beacon probe, as described inExample 1.

Real-time PCR data are provided below (Table 20) for PCR reactionscontaining C. albicans genomic DNA prepared from C. albicans lysateusing the indicated number of cells/mL. The reactions were set up usinglysates prepared over multiple days not the same sample lysate detectedover multiple days, thus the variation in the entire process is shown.As shown in Table 20 and FIG. 8A, C. albicans was successfully detectedusing the C. albicans specific molecular beacon probe, andsemi-quantitative detection of C. albicans was possible using as few as˜3 cells/mL.

TABLE 20 C. albicans cells/mL 520 400 280 52 40 28 5.2 4 2.8 operatordate 2.716003 2.6021 2.44716 1.716 1.6021 1.4472 0.699 0.6021 0.447158030 NP May 1, 2012 28.47 32.83 ND 28.45 32.95 ND 28.45 32.89 ND 28.4732.86 ND NP Mar. 20, 2012 28.59 32.15 34.84 ND 28.68 32.1 34.86 ND 28.6632.02 34.66 ND 28.68 32.02 34.86 ND NP Mar. 30, 2012 28.36 28.75 29.1531.64 31.81 32.42 34.03 34.66 ND 28.35 28.79 29.18 31.67 31.73 32.4434.03 33.76 34.77 ND 28.45 28.77 29.24 31.64 31.78 32.5 34.12 34.09 ND28.29 28.7 31.9 31.79 33.97 34.18 ND NP Mar. 28, 2012 28.48 28.77 29.1531.83 31.8 32.69 34.88 34.54 34.99 ND 28.5 28.8 29.16 31.62 31.68 32.6634.57 34.21 34.94 ND 28.32 28.78 29.16 31.56 31.7 32.44 34.77 34.53 34.9ND 28.28 28.78 29.15 31.71 31.73 32.78 34.32 34.55 34.81 ND NP Apr. 19,2012 28.14 28.65 29.25 31.8 31.75 32.59 34.08 34.18 34.29 ND 28.12 28.5729.28 31.75 31.84 32.63 33.98 34.03 34.42 ND 28.03 27.24 29.25 31.5831.64 32.58 33.9 34.23 34.55 ND 28.11 28.64 29.21 31.61 31.72 32.4934.23 34.31 ND mean 28.28583 28.585 29.1982 31.693 32.04 32.565 34.26234.361 34.664 sd 0.155649 0.3384 0.04916 0.1068 0.4547 0.1175 0.35610.3356 0.26000855 cv 0.55% 1.18% 0.17% 0.34% 1.42% 0.36% 1.04% 0.98%0.75%

The above data demonstrate that the C. albicans molecular beacon probecan be used to successfully detect C. albican using either purifiedgenomic DNA as an input in a real-time PCR assay or, alternatively,using a fungal cell lysate as an input in the assay.

Example 9: Detection of C. glabrata Using a Species-Specific MolecularBeacon Probe in a Real-Time PCR Assay

A HEX labeled C. glabrata probe (having the sequence of SEQ ID NO: 3)was used at 300 nM concentration in a real-time PCR assay that wasperformed according to the parameters described in Table 1 above. 100 μlof diluted genomic DNA was added to the PCR reaction mix and 45 cyclesof amplification were performed with an annealing temperature of 60° C.A titration using different amounts of genomic DNA template wasperformed. Real-time PCR data are provided in Table 21 for reactionscontaining 0 to 50,000 copies (i.e., 0 (no template), 5, 50, 500, 5,000,or 50,000) of genomic DNA per reaction. Four replicates for eachreaction condition were performed and the average Cp values werecalculated. The average amplification efficiency (2.23) and the PCRefficiency (123%) for the reactions were also calculated. Table 21 showsthat the C. glabrata molecular beacon probe was used successfully todetect the target nucleic acid molecule using genomic DNA isolated fromC. glabrata.

TABLE 21 C. glabrata detection in singleplex reactions Std copies/rx LogCp1 Cp2 Cp3 Cp4 Average Cp CV 50000 5 23.72 23.78 23.68 23.71 23.72 0.040.2% 5000 4 27.1 27.18 27.22 27.24 27.19 0.06 0.2% 500 3 30.56 30.6730.64 30.51 30.60 0.07 0.2% 50 2 33.17 33.46 33.44 33.55 33.41 0.16 0.5%5 1 35 35 35 35 35.00 0.00 0.0% no ND ND ND ND template slope −2.87 0.35amplification 2.23 efficiency = PCR efficiency = 123%

Next, a similar assay was performed using a C. glabrata lysate ratherthan genomic DNA according to the assay described in Example 8.

Real-time PCR data are provided below (Table 22) for PCR reactionscontaining C. glabrata genomic DNA prepared from C. glabrata lysateusing the indicated number of cells/mL. The reactions were set up usinglysates prepared over multiple days not the same sample lysate detectedover multiple days, thus the variation in the entire process is shown.As shown in Table 22 and FIG. 8B, C. glabrata was successfully detectedusing the C. glabrata specific molecular beacon probe, andsemi-quantitative detection of C. glabrata was possible using as few as˜3 cells/mL.

TABLE 22 C. glabrata cells/mL 520 400 280 52 40 28 5.2 4 2.8 operatodate 2.716003 2.60206 2.447158 1.716003 1.60205 1.447158 0.69897 0.602060.447158 0 NP Mar. 29, 2012 28.45 28.75 29.05 31.77 31.91 32.39 34.2734.53 34.71 ND 28.55 28.76 28.94 81.86 31.95 32.39 34.07 34.54 34.97 ND28.3 28.59 29.09 31.98 32.31 34.08 34.67 34.86 ND 28.51 29.68 31.8532.58 34.81 ND 28.93 29.05 29.44 31.57 31 32.23 32.76 33.45 34.24 ND28.82 28.95 29.35 31.59 31.68 32.46 33.92 33.65 34.94 ND 28.83 28.9129.29 31.54 31.95 32.17 34.27 34.38 ND NP May 10, 2012 29.86 32.83 ND29.25 32.95 ND 29.75 32.89 ND 29.74 32.86 ND CW May 10, 2012 29.65 32.98ND 29.75 32.88 ND 29.79 33.03 ND 29.74 33.06 ND NP Mar. 20, 2012 28.6935.19 35.19 ND 28.81 32.18 35.42 ND 28.84 32.55 35.24 ND mean 28.6257128.83667 29.26286 31.71333 31.745 32.36143 33.895 34.168 34.70143 sd0.231722 0.168721 0.255976 0.137502 0.380986 0.138856 0.57183 0.5712440.283515 cv 0.81% 0.59% 0.87% 0.43% 1.20% 0.43% 1.69% 1.67% 0.82%

The above data demonstrate that the C. glabrata molecular beacon probecan be used to successfully detect C. glabrata using either purifiedgenomic DNA as an input in a real-time PCR assay or, alternatively,using a fungal cell lysate as an input in the assay.

Example 10: Detection of C. krusei Using a Species-Specific MolecularBeacon Probe in a Real-Time PCR Assay

A HEX labeled C. krusei probe (having the sequence of SEQ ID NO: 6) wasused at 300 nM concentration in a real-time PCR assay that was performedaccording to the parameters described in Table 1 above. 100 μl ofdiluted genomic DNA was added to the PCR reaction mix and 45 cycles ofamplification were performed with an annealing temperature of 60° C. Atitration using different amounts of genomic DNA template was performed.Real-time PCR data are provided in Table 23 for reactions containing 0to 50,000 copies (i.e., 0 (no template), 5, 50, 500, 5,000, or 50,000)of genomic DNA per reaction. Four replicates for each reaction conditionwere performed and the average Cp values were calculated. The averageamplification efficiency (2.05) and the PCR efficiency (105%) for thereactions were also calculated. Table 23 shows that the C. kruseimolecular beacon probe was used successfully to detect the targetnucleic acid molecule using genomic DNA isolated from C. krusei.

TABLE 23 C. krusei detection in singleplex reactions Std copies/rx LogCp1 Cp2 Cp3 Cp4 Average Cp CV 50000 5 22.17 22.3 22.15 22.21 0.08 0.4%5000 4 25.77 25.86 25.75 25.85 25.81 0.06 0.2% 500 3 29.15 29.03 29.2129.2 29.15 0.08 0.3% 50 2 32.17 32.25 32.2 32.34 32.24 0.07 0.2% 5 1 3535 35 35 35.00 0.00 0.0% no ND ND ND ND template slope −3.2019 0.31amplification 2.05 efficiency = PCR efficiency = 105%

Next, a similar assay was performed using a C. krusei lysate rather thangenomic DNA according to the assay described in Example 8.

Real-time PCR data are provided below (Table 24) for PCR reactionscontaining C. krusei genomic DNA prepared from C. krusei lysate usingthe indicated number of cells/mL. The reactions were set up usinglysates prepared over multiple days not the same sample lysate detectedover multiple days, thus the variation in the entire process is shown.As shown in Table 24 and FIG. 8C, C. krusei was successfully detectedusing the C. krusei specific molecular beacon probe, andsemi-quantitative detection of C. krusei was possible using as few as ˜3cells/mL.

TABLE 24 C. krusei cell mL 520 400 280 52 40 28 5.2 4 2.8 operator date2.716003 2.60206 2.447158 1.716003 1.60205 1.447158 0.69897 0.602060.447158 0 NP Mar. 21, 2012 27.81 31.21 34.72 ND 27.59 31.1 34.88 ND27.83 31.17 34.91 ND 27.77 13.12 34.94 ND NP Mar. 23, 2012 28.71 31.934.81 ND 28.83 31.92 34.97 ND 28.75 31.85 34.88 ND 28.69 31.32 32.8 NDNP Mar. 30, 2012 27.85 28.01 28.43 31.19 31.53 31.67 33.47 34.34 34.28ND 27.77 28.03 28.36 30.88 31.51 31.69 33.66 34.86 34.16 ND 27.84 27.9628.36 30.84 31.51 31.74 33.2 34.59 34.43 ND 27.24 27.98 30.82 31.2633.95 34.34 ND 27.18 27.96 30.76 31.25 33.92 34.13 ND 27.77 22.88 30.8531.46 33.69 33.96 ND 27.32 27.9 30.79 31.24 33.78 34.5 ND NP May 10,2014 28.2 31.75 ND 28.22 31.72 ND 28.2 31.74 ND 28.35 31.75 ND CW May10, 2012 28.48 31.59 ND 28.45 31.73 ND 28.37 31.7 ND 28.56 31.6 ND NPMar. 20, 2012 27.74 30.92 34.84 ND 27.82 31.07 34.72 ND 27.91 31.2234.52 ND mean 27.49571 28.14316 28.38333 30.87571 31.42333 31.7 33.4433334.402667 34.25714 sd 0.307184 0.364662 0.040415 0.14409 0.2791480.036056 0.231157 0.6403399 0.187146 cv 1.12% 1.30% 0.14% 0.47% 0.89%0.11% 0.69% 1.86% 0.55%

The above data demonstrate that the C. krusei molecular beacon probe canbe used to successfully detect C. krusei using either purified genomicDNA as an input in a real-time PCR assay or, alternatively, using afungal cell lysate as an input in the assay.

Example 11: Detection of C. parapsilosis Using a Species-SpecificMolecular Beacon Probe in a Real-Time PCR Assay

A HEX labeled C. parapsilosis probe (having the sequence of SEQ ID NO:5) was used at 300 nM concentration in a real-time PCR assay that wasperformed according to the parameters described in Table 1 above. 100 μlof diluted genomic DNA was added to the PCR reaction mix and 45 cyclesof amplification were performed with an annealing temperature of 60° C.A titration using different amounts of genomic DNA template wasperformed. Real-time PCR data are provided in Table 25 for reactionscontaining 0 to 50,000 copies (i.e., 0 (no template), 5, 50, 500, 5,000,or 50,000) of genomic DNA per reaction. Four replicates for eachreaction condition were performed and the average Cp values werecalculated. The average amplification efficiency (2.10) and the PCRefficiency (110%) for the reactions were also calculated. Table 25 showsthat the C. parapsilosis molecular beacon probe was used successfully todetect the target nucleic acid molecule using genomic DNA isolated fromCandida parapsilosis.

TABLE 25 C. parapsilosis detection in singleplex rx Std copies/rx LogCp1 Cp2 Cp3 Cp4 Average Cp CV 50000 5 22.55 22.43 22.47 22.45 22.48 0.050.2% 5000 4 25.91 25.85 25.9 25.72 25.85 0.09 0.3% 500 3 29.2 29.2 29.2529.25 29.23 0.03 0.1% 50 2 32.13 32.16 32.15 32.31 32.19 0.08 0.3% 5 135 35 34.53 35 34.84 0.24 0.7% no ND ND ND ND template slope −3.10790.32 amplification 2.10 efficiency = PCR efficiency = 110%

Next, a similar assay was performed using a C. parapsilosis lysaterather than genomic DNA according to the assay described in Example 8.

Real-time PCR data are provided below (Table 26) for PCR reactionscontaining C. parapsilosis genomic DNA prepared from C. parapsilosislysate using the indicated number of cells/mL. The reactions were set upusing lysates prepared over multiple days not the same sample lysatedetected over multiple days, thus the variation in the entire process isshown. As shown in Table 26 and FIG. 8D, C. parapsilosis wassuccessfully detected using the C. parapsilosis specific molecularbeacon probe, and semi-quantitative detection of C. parapsilosis waspossible using as few as ˜3 cells/mL.

TABLE 26 C. parapsilosis cells/ op- 520 400 280 52 40 28 5.2 4 2.8 0erator date 2.716003 2.60206 2.447158 1.716003 1.60206 1.447158 0.698970.60206 0.447158 0 NP Mar. 23, 2012 28.71 31.99 35.06 ND 28.71 31.9334.89 ND 28.63 31.89 35.25 ND NP Apr. 2, 2012 28.3 28.59 29.02 31.331.36 31.67 33.37 33.27 33.49 ND 28.24 28.55 29.03 31.33 31.16 31.7433.1 33.22 33.47 ND 28.28 28.6 29.05 31.32 31.23 31.65 33.17 33.19 33.17ND 28.21 28.52 29.06 31.2 31.14 31.65 32.92 33.06 33.3 ND 28.05 28.2828.8 29.49 30.75 29.88 29.11 32.18 32.01 ND 28.05 28.3 28.77 30.81 30.6130.72 32.22 32.16 32.43 ND 28.06 28.16 28.88 30.86 30.53 31.25 31.6432.03 31.47 ND NP Apr. 30, 2012 28.9 31.19 ND 28.94 31.45 ND 28.9 31.21ND 28.93 31.49 ND NP May 11, 2012 28.66 31.07 ND 28.68 31.12 ND 28.731.11 ND 28.67 31.11 ND CW May 11. 2012 28.94 31.17 ND 29.02 31.2 ND28.96 31.23 ND 29.01 31.24 ND mean 28.2575 28.68333 29.04 31.287531.93667 31.6775 33.14 33.99143 33.3575 sd 0.040311 0.046188 0.0182570.059652 0.050332 0.04272 0.186011 1.013138 0.1513 cv 0.14% 0.16% 0.06%0.19% 0.16% 0.13% 0.56% 2.98% 0.45%

The above data demonstrate that the C. parapsilosis molecular beaconprobe can be used to successfully detect C. parapsilosis using eitherpurified genomic DNA as an input in a real-time PCR assay or,alternatively, using a fungal cell lysate as an input in the assay.

Example 12: Detection of C. tropicalis Using a Species-SpecificMolecular Beacon Probe in a Real-Time PCR Assay

A HEX labeled C. tropicalis probe (having the sequence of SEQ ID NO: 2)was used at 300 nM concentration in a real-time PCR assay that wasperformed according to the parameters described in Table 1 above. 100 μlof diluted genomic DNA was added to the PCR reaction mix and 45 cyclesof amplification were performed with an annealing temperature of 60° C.A titration using different amounts of genomic DNA template wasperformed. Real-time PCR data are provided in Table 27 for reactionscontaining 0 to 50,000 copies (i.e., 0 (no template), 5, 50, 500, 5,000,or 50,000) of genomic DNA per reaction. Four replicates for eachreaction condition were performed and the average Cp values werecalculated. The average amplification efficiency (2.11) and the PCRefficiency (111%) for the reactions were also calculated. Table 27 showsthat the C. tropicalis molecular beacon probe was used successfully todetect the target nucleic acid molecule using genomic DNA isolated fromC. tropicalis.

TABLE 27 C. tropicalis detection in singleplex rx Std copies/rx Log Cp1Cp2 Cp3 Cp4 Average Cp CV 50000 5 22.81 22.81 22.23 22.72 22.64 0.281.2% 5000 4 26.23 26.08 26.23 26.13 26.17 0.08 0.3% 500 3 29.35 29.3729.31 29.31 29.34 0.03 0.1% 50 2 32.59 32.26 32.14 32.34 32.33 0.19 0.6%5 1 35 35 35 35 35.00 0.00 0.0% no ND ND ND ND template slope −3.09 0.32amplification 2.11 efficiency = PCR efficiency = 111%

Next, a similar assay was performed using a C. tropicalis lysate ratherthan genomic DNA according to the assay described in Example 8.

Real-time PCR data are provided below (Table 28) for PCR reactionscontaining C. tropicalis genomic DNA prepared from C. tropicalis lysateusing the indicated number of cells/mL. The reactions were set up usinglysates prepared over multiple days not the same sample lysate detectedover multiple days, thus the variation in the entire process is shown.As shown in Table 28 and FIG. 8E, C. tropicalis was successfullydetected using the C. tropicalis specific molecular beacon probe, andsemi-quantitative detection of C. tropicalis was possible using as fewas ˜3 cells/mL.

TABLE 28 C. tropicalis cells/mL 520 400 280 52 40 28 5.2 4 2.8 operatordate 2.716003 2.60206 2.447158 1.716003 1.60206 1.447158 0.69897 0.602060.447158 0 NP Apr. 30, 2012 28.94 31.1 ND 28.89 30.98 ND 28.96 31.04 ND28.92 30.91 ND NP Mar. 23, 2012 28.71 31.9 34.81 ND 28.83 31.92 34.97 ND28.75 31.85 34.88 ND NP Apr. 3, 2012 28.69 31.32 32.8 ND 28.36 28.7229.15 31.64 31.28 32.42 32.81 34.66 ND 28.35 28.65 29.18 31.67 31.4232.44 34.03 32.68 34.77 ND 28.45 28.69 29.24 31.64 31.35 32.5 34.12 32.8ND NP Apr. 4, 2012 27.87 28.58 28.88 30.49 30.53 31 32.22 32.04 32.73 ND27.85 28.56 29.02 30.5 30.77 31.06 31.83 31.96 32.77 ND 27.82 28.5428.99 30.43 30.67 31.24 31.99 32.84 ND NP May 11, 2012 28.61 30.93 ND28.68 30.84 ND 28.66 30.96 ND 28.65 30.97 ND CW May 11, 2012 28.87 30.86ND 28.93 30.96 ND 28.92 30.98 ND 28.98 31.02 ND NP Mar. 21, 2012 28.5731.99 35.07 ND 28.67 31.92 34.57 ND 28.59 32.06 34.55 ND 28.61 32.0534.46 ND mean 28.11667 28.73158 29.07667 31.06167 31.152222 31.7766732.838 33.30556 33.554 sd 0.298239 0.132173 0.135745 0.645025 0.41140440.745913 1.138143 1.228455 1.061287 cv 1.06% 0.46% 0.47% 2.08% 1.32%2.35% 3.47% 3.69% 3.16%

The above data demonstrate that the C. tropicalis molecular beacon probecan be used to successfully detect C. tropicalis using either purifiedgenomic DNA as an input in a real-time PR assay or, alternatively, usinga fungal cell lysate as an input in the assay.

Example 13: Detection of Candida albicans in a Multiplex Reaction withCandida albicans, Candida Krusei, and Candida glabrata

The molecular beacon probes can also be used to successfully detectCandida target nucleic acid molecules in a multiplexed reaction thatincludes nucleic acid molecules from two or more Candida species.

A multiplex reaction was performed using nucleic acid molecules (genomicDNA) from C. albicans, C. krusei, and C. glabrata. 300 nM of eachCandida species-specific beacon probe (corresponding to SEQ ID NOs: 1,6, and 3, respectively; 0.3 uL per 100 uL reaction) were used in areal-time PCR assay that was performed according to the parametersdescribed in Table 1 above. The C. albicans, C. krusei, and C. glabratamolecular beacons are each labeled with spectrally distinguishablefluororphors that are detected in their respective channels. 100 μl ofdiluted genomic DNA was added to the PCR reaction mix and 45 cycles ofamplification were performed with an annealing temperature of 60° C. Atitration using different amounts of genomic DNA template was performed.Real-time PCR data are provided in Table 29 for reactions containing 0to 50,000 copies (i.e., 0 (no template), 5, 50, 500, 5,000, or 50,000)of genomic DNA per reaction. Four replicates for each reaction conditionwere performed and the average Cp values were calculated. The averageamplification efficiency (2.00) and the PCR efficiency (100%) for thereactions were also calculated. Table 29 shows that the C. albicansmolecular beacon probe was used successfully to detect the C. albicanstarget nucleic acid molecules using genomic DNA isolated from C.albicans, even in the presence C. krusei and C. glabrata nucleic acidmolecules, as well as molecular beacons for all of these Candidaspecies. Notably, the amplification efficiency and PCR efficiency in themultiplex reaction was similar to that observed in the singleplexreaction (see Example 8 and Table 19). Thus, the limit of detectionusing the molecular beacon probes is not changing even when they areused in combination. Similarly, the presence of multiple probes in thesample is not affecting the efficiency of the nucleic acid amplificationreaction.

TABLE 29 C. albicans detection in multiplex rx (C. albicans, C. krusei,and C. glabrata multiplex) copies/rx Cp1 Cp2 Cp3 Cp4 Average Std Cp CV50000 21.69 21.76 21.05 21.68 21.55 0.33 1.5% 5000 25.1 25.14 25.0825.02 25.09 0.05 0.2% 500 28.7 28.55 28.51 28.52 28.57 0.09 0.3% 5031.68 31.77 31.92 31.79 0.12 0.4% 5 34.45 35 34.93 35 34.79 0.27 0.8% notemplate ND ND ND ND slope −3.32 0.30 amplification 2.00 efficiency =PCR efficiency = 100%

Example 14: Detection of C. glabrata in a Multiplex Reaction with C.albicans, C. krusei, and C. glabrata

A molecular beacon probe for C. glabrata (SEQ ID NO: 3) can be used tosuccessfully detect C. glabrata nucleic acid molecules in a multiplexreaction that also includes nucleic acid molecules from C. albicans andC. krusei. The multiplex reaction was performed as described in Example13. Real-time PCR data are provided in Table 30 for reactions containing0 to 50,000 copies (i.e., 0 (no template), 5, 50, 500, 5,000, or 50,000)of genomic. DNA per reaction. Four replicates for each reactioncondition were performed and the average Cp values were calculated. Theaverage amplification efficiency (2.02) and the PCR efficiency (102%)for the reactions were also calculated. Table 30 shows that the C.glabrata molecular beacon probe was used successfully to detect C.glabrata target nucleic acid molecules using genomic DNA isolated fromC. glabrata, even in the presence C. albicans and C. krusei nucleic acidmolecules, as well as molecular beacons for all of these Candidaspecies. Notably, the amplification efficiency and PCR efficiency in themultiplex reaction was similar to that observed in the singleplexreaction (see Example 9 and Table 21).

TABLE 30 C. glabrata detection in multiplex rx (C. albicans, C. krusei,and C. glabrata multiplex) copies/rx Cp1 Cp2 Cp3 Cp4 Average Std Cp CV50000 23.65 23.79 23.25 23.75 23.61 0.25 1.0% 5000 27.08 27.12 26.9227.03 27.04 0.09 0.3% 500 30.55 30.49 30.54 30.53 0.03 0.1% 50 33.2433.3 33.49 33.38 33.35 0.11 0.3% 5 35 35 35.00 0.00 0.0% no template NDND ND ND slope −3.27 0.31 amplification 2.02 efficiency = PCR efficiency= 102%

Example 15: Detection of C. krusei in a Multiplex Reaction with C.albicans, C. krusei, and C. glabrata

A molecular beacon probe for Candida krusei (SEQ ID NO: 6) can be usedto successfully detect C. krusei nucleic acid molecules in a multiplexreaction that also includes nucleic acid molecules from C. albicans andC. glabrata. The multiplex reaction was performed as described inExample 13. Real-time PCR data are provided in Table 26 for reactionscontaining 0 to 50,000 copies (i.e., 0 (no template), 5, 50, 500, 5,000,or 50,000) of genomic DNA per reaction. Four replicates for eachreaction condition were performed and the average Cp values werecalculated. The average amplification efficiency (2.12) and the PCRefficiency (112%) for the reactions were also calculated. Table 31 showsthat the C. krusei molecular beacon probe was used successfully todetect C. krusei target nucleic acid molecules using genomic DNAisolated from C. krusei, even in the presence C. albicans and C.glabrata nucleic acid molecules, as well as molecular beacons for all ofthese Candida species. Notably, the amplification efficiency and PCRefficiency in the multiplex reaction was similar to that observed in thesingleplex reaction (see Example 10 and Table 23).

TABLE 31 C. krusei detection in multiplex rx (C. albicans, C. krusei,and C. glabrata multiplex) copies/rx Cp1 Cp2 Cp3 Cp4 Average Std Cp CV50000 22.18 22.34 22.12 22.24 22.22 0.09 0.4% 5000 25.74 25.66 25.6825.69 0.04 0.2% 500 29.13 29.26 29.38 28.93 29.18 0.19 0.7% 50 32.1432.09 32.18 32.22 32.16 0.06 0.2% 5 34.36 33.68 35 35 34.35 0.63 1.8% notemplate ND ND ND ND slope −3.07 0.33 2.12 112%

Example 16: Detection of C. parapsilosis in a Multiplex Reaction with C.parapsilosis and C. tropicalis

A molecular beacon probe for Candida parapsilosis (SEQ ID NO: 4) can beused to successfully detect C. parapsilosis nucleic acid molecules in amultiplex reaction that also includes nucleic acid molecules from C.tropicalis. The multiplex reaction was performed using nucleic acidmolecules (genomic DNA) from C. parapsilosis and C. tropicalis. 300 nMof each Candida species-specific beacon probe (corresponding to SEQ IDNOs: 4 and 2, respectively; 0.3 uL per 100 uL reaction) were used in areal-time PCR assay that was performed according to the parametersdescribed in Table 1 above. 100 μl of diluted genomic DNA was added tothe PCR reaction mix and 45 cycles of amplification were performed withan annealing temperature of 60° C. A titration using different amountsof genomic DNA template was performed. Real-time PCR data are providedin Table 32 for reactions containing 0 to 50,000 copies (i.e., 0 (notemplate), 5, 50, 500, 5,000, or 50,000) of genomic DNA per reaction.Four replicates for each reaction condition were performed and theaverage Cp values were calculated. The average amplification efficiency(2.07) and the PCR efficiency (107%) for the reactions were alsocalculated. Table 32 shows that the C. parapsilosis molecular beaconprobe was used successfully to detect C. parapsilosis target nucleicacid molecules using genomic DNA isolated from C. parapsilosis, even inthe presence C. tropicalis nucleic acid molecules, as well as molecularbeacons for both of these Candida species. Notably, the amplificationefficiency and PCR efficiency in the multiplex reaction was similar tothat observed in the singleplex reaction (see Example 11 and Table 25).

TABLE 32 C. parapsilosis detection in multiplex rx (C. tropicalis, C.parpasilosis) copies/rx Cp1 Cp2 Cp3 Cp4 Average Std Cp CV 50000 22.0322.33 22.47 22.47 22.33 0.21 0.9% 5000 25.83 25.86 25.81 25.81 25.830.02 0.1% 500 29.17 29.16 29.18 29.13 29.16 0.02 0.1% 50 32.03 32.1132.29 32.1 32.13 0.11 0.3% 5 35 35 35 35 35.00 0.00 0.0% no template NDND ND ND slope 3.17 0.32 2.07 107%

Example 17: Detection of C. tropicalis in a Multiplex Reaction with C.parapsilosis and C. tropicalis

A molecular beacon probe for C. tropicalis (SEQ ID NO: 2) can be used tosuccessfully detect C. tropicalis nucleic acid molecules in a multiplexreaction that also includes nucleic acid molecules from C. parapsilosis.The multiplex reaction was performed as described in Example 16.Real-time PCR data are provided in Table 33 for reactions containing 0to 50,000 copies (i.e., 0 (no template), 5, 50, 500, 5,000, or 50,000)of genomic DNA per reaction. Four replicates for each reaction conditionwere performed and the average Cp values were calculated. The averageamplification efficiency (2.11) and the PCR efficiency (111%) for thereactions were also calculated. Table 33 shows that the C. tropicalismolecular beacon probe was used successfully to detect Candidatropicalis target nucleic acid molecules using genomic DNA isolated fromC. tropicalis, even in the presence C. parapsilosis nucleic acidmolecules, as well as molecular beacons for both of these Candidaspecies. Notably, the amplification efficiency and PCR efficiency in themultiplex reaction was similar to that observed in the singleplexreaction (see Example 12 and Table 27).

TABLE 33 C. tropicalis detection in multiplex rx (C. tropicalis, C.parpasilosis) copies/rx Cp1 Cp2 Cp3 Cp4 Average Std Cp CV 50000 22.7522.75 22.94 22.43 22.72 0.21 0.9% 5000 26.1 26.05 26.33 26.09 26.14 0.130.5% 500 29.19 29.45 29.51 29.29 29.36 0.15 0.5% 50 32.26 32.24 32.6932.15 32.34 0.24 0.7% 5 35 35 35 35 35.00 0.00 0.0% no template ND ND NDND slope −3.08 0.33 2.11 111%

Example 18: Detection of Candida Species Using a Beat Denatured FungalLysate

To determine if the molecular beacon probes could be used for thedetection of Candida cells directly using a 95° C. heat denaturationstep to lyse the Candida cells instead of mechanical lysis, such as bybead beating, we spiked 50 uL of TE containing Coulter counted Candidacells directly into a 96-well plate. We then added the molecular beaconprobe-containing master mix on top of the cells and conducted an RT-PCRusing the parameters described in Example 1 above.

Real-time PCR data are provided below (Tables 34-38) for PCR reactions,each of which contains C. albicans, C. glabrata, C. krusei, C.parapsilosis, and C. tropicalis genomic DNA prepared from heat denaturedlysates containing the indicated number of cells. As shown in Tables34-38, the Candida specific molecular beacon probes (C. albicans: SEQ IDNO: 1; C. glabrata: SEQ ID NO: 3; C. krusei: SEQ ID NO: 6; C.parapsilosis: SEQ ID NO: 4; and C. tropicalis: SEQ ID NO: 2)successfully detected the indicated Candida nucleic acid moleculesfollowing lysis by heat denaturation. Detection was possible even up to10 cells.

TABLE 34 Detection of C. albicans in fungal lysate C. albicans cells Cp1Cp2 Cp3 Average Std Cp CV 1000 37.18 36.78 37.4 37.12 0.31 0.8% 100 ND40 40 40.00 0.00 0.0% 50 40 39.09 ND 39.55 0.64 1.6% 10 40 ND ND 40.00no template ND ND ND

TABLE 35 Detection of C. glabrata in fungal lysate C. glabrata cells Cp1Cp2 Cp3 Average Std Cp CV 1000 34 33.99 34.31 34.10 0.18 0.5% 100 37.6136.63 36.26 36.83 0.70 1.9% 50 40 36.85 39.43 38.76 1.68 4.3% 10 ND 4040 40.00 0.00 0.0% no template ND ND ND

TABLE 36 Detection of C. krusei in fungal lysate C. krusei cells Cp1 Cp2Cp3 Average Std Cp CV 1000 29.53 28.98 29.00 29.17 0.31 1.1% 100 32.5132.54 32.26 32.44 0.15 0.5% 50 33.28 33.48 33.54 33.43 0.14 0.4% 1035.74 35.76 36.11 35.87 0.21 0.6% no template ND ND ND

TABLE 37 Detection of C. parapsilosis in fungal lysate C. parapsilosiscells Cp1 Cp2 Cp3 Average Std Cp CV 1000 36.16 35.63 35.35 35.71 0.411.2% 100 37.78 38.59 39.19 38.52 0.71 1.8% 50 39.54 40 38.28 39.27 0.892.3% 10 ND 40 ND no template ND ND ND

TABLE 38 Detection of C. tropicalis in fungal lysate C. tropicalis cellsCp1 Cp2 Cp3 Average Std Cp CV 1000 32.93 33.37 33.2 33.17 0.22 0.7% 10036.03 36.23 36.82 36.36 0.41 1.1% 50 37.14 37.74 37.58 37.49 0.31 0.8%10 40 40 40 40.00 0.00 0.0% no template ND 40 ND

As positive controls, the same Candida nucleic acid molecules preparedvia the PCR described above were detected using Candida specific probes(C. albicans: SEQ ID NOs: 13 and 14; C. glabrata: SEQ ID NOs: 17 and 18;C. krusei: SEQ ID NOs: 15 and 16; C. parapsilosis: SEQ ID NOs: 21 and22; and C. tropicalis: SEQ ID NOs: 19 and 20) conjugated to magneticnanoparticles for use in an NMR based assay, as is described above. Inshort, the hybridization induced agglomeration assays is performed byaliquoting 15 μL of the amplification reaction into 0.2 mL thin walledPCR tubes and incubating within a sodium phosphate hybridization buffer(4×SSPE) with the indicated pairs of oligonucleotide probe derivatizednanoparticles at a final iron concentration of 0.2 mM iron per reaction.Hybridization reactions were incubated for 3 minutes at 95° C. followedby 30 minutes incubation at 60° C. within a shaking-incubator set at anagitation speed of 1000 rpm (Vortemp, LabNet International). Hybridizedsamples are then placed in a 37° C. heating block to equilibrate thetemperature to that of the MR reader for 3 minutes. Each sample is thensubjected to a 5 second vortexing step (3000 rpm) and inserted into theMR reader for T2 measurement. The baseline T2 signal is ˜30-40 msec,thus a signal<45 indicates no target DNA is present.

As shown in Tables 39-43, there is 97% concordance between the detectedCandida cells via the molecular beacon probes and the T2 detectionreactions run on the RT-PCR generated amplicons. We observe one instancein which C. glabrata nucleic acid molecules from the sample preparedusing cells was not detected via RT-PCR but was detected with theNMR-based T2 assay (compare Cp1 in Table 35 to Rep1 in Table 40), andone sample in which C. tropicalis nucleic acid molecules from the sampleprepared using 10 cells was detected via RT-PCR but not by the NMR-basedT2 assay (compare Cp1 in Table 38 to Rep1 in Table 43). These discordantresults are highlighted in bold and italics in the tables below.

TABLE 39 C. albicans RT-PCR product, T2 detection Rep1 Rep2 Rep3 AverageStd Cp CV 253.8 239.06 253.78 248.88 8.50 3.4% 36.09 223.18 250.38236.78 19.23 8.1% 251.33 233.13 36.72 242.23 0.0% 220.19 36.62 36.17220.19 0.0% 36.91 36.58 38.53 37.34 1.04 2.8%

TABLE 40 C. glabrata RT-PCR product, T2 detection Std Rep1 Rep2 Rep3Average Cp CV 102.97 98.49 96.11 99.19 3.48 3.5% 117.8 121.86 112.59117.42 4.65 4.0% 125.25 108.89 180.44 138.19 37.49 27.1% 273.68 139.8189.81 201.10 67.65 33.6% 35.16 35.19 36.64 35.66 0.85 2.4%

TABLE 41 C. krusei RT-PCR product, T2 detection Rep1 Rep2 Rep3 AverageStd Cp CV 105.92 116.9 104.72 109.18 6.71 6.1% 106.36 118.04 107.04110.48 6.56 5.9% 110.11 119.5 109.77 113.13 5.52 4.9% 122.32 128.94119.36 123.54 4.91 4.0% 35.63 35.26 37.55 36.15 1.23 3.4%

TABLE 42 C. parapsilosis RT-PCR product, T2 detection Rep1 Rep2 Rep3Average Std Cp CV 303.44 307.97 279.39 296.93 15.36  5.2% 343.76 327.83361.66 344.42 16.92  4.9% 390.33 464.65 315.78 390.25 74.44 19.1% 32.27448.74 32.66 31.98 32.02 32.94 32.31 0.54  1.7%

TABLE 43 C. tropicalis RT-PCR product, T2 detection Rep1 Rep2 Rep3Average Std Cp CV 279.39 471.34 441.24 397.3233 103.2362 26.0% 361.66438.41 435.62 411.90 43.53 10.6% 315.78 452.44 436.73 401.65 74.78 18.6%32.66 475.71 530.58 346.32 273.02 78.8% 36.04 35.07 35.36 35.49 0.501.4%

Example 19: Detection of Candida Species Using Melt-Curve Analysis

Because the Tm of the Candida species-specific molecular beacon probesdiffer by at least 1 degree, it is possible to multiplex the beaconprobes with the same fluorophore and deconvolute the signals using meltcurve analysis alone. Molecular beacon probes for C. albicans (SEQ IDNO: 1), C. glabrata (SEQ ID NO: 3), C. krusei (SEQ ID NO: 6), C.parapsilosis (SEQ ID NO: 4), and C. tropicalis (SEQ ID NO: 2) were alllabeled with a HEX fluorophore and added at 300 nm concentration to asingle sample that included target nucleic acid molecules for each ofthe Candida species.

As shown in FIGS. 9A and 9B, the presence of target nucleic acidmolecules for the indicated Candida species targets was determinedwithin the sample simply by looking for the presence of thespecies-specific melt curve and using the Tm as a means of speciesidentification. Use of the melt-curve analysis abrogates the need to usemultiple different fluorophores in a single multiplexed reaction andsimplifies the instrument design. It should be noted that there is nomelt curve when the target nucleic acid molecule is not present in thereaction. The Tm is the temperature at which a decrease in fluorescenceis observed and this only occurs when the hybridized beacon probe ismelted off the target and the beacon probe stem-loop structure reforms.

Example 20: Molecular Beacon Analysis Using a Post-Blood Culture

To perform a blood culture analysis, up to 10 ml of a blood sample froma patient suspected of having a blood systemic infection are incubatedin a blood culture bottle. The blood culture media typically arecomposed to promote the growth of either aerobic or anaerobic pathogens.Depending on the specific requirements for growth of the suspectedpathogen, culture media is composed of a number of nutrients that feedpathogens and resins that absorb antibiotics/antifungals that might bepresent in the blood specimen and inhibit growth. When a pathogenreaches a concentration of 10⁶ to 10⁸ CFU/mL, it produces enough carbondioxide through its normal metabolic pathways to activate the bloodculture detection system, thus signaling a positive blood culture forthe original sample. At this time, a sample from this culture bottle isaliquoted onto a species-specific detection methodology, which may be aculture plate or another system that identifies the pathogenic speciesand assesses its susceptibility to antimicrobials (e.g., Vitek 2,Biomeriuex, Durham, N.C.). Species identification is typically performedover a day, although it can be significantly longer.

In order to expedite the species identification of Candida that may bepresent in a blood culture, a singleplex or multiplex reaction fordetecting one or more Candida species, according to the methodsdescribed in, e.g., Examples 1-19 above, can be used. The protocolinvolves adding a sample from the positive blood culture bottle to thesingleplex or multiplex reaction, centrifuging the sample to pellet thepathogen cells, which may be optionally washed one or more times toremove interferents from the blood culture media, lysing the cells andusing the nucleic acid molecules from the cells in a molecular beaconprotocol described in one or more of Examples 1-19 above. This processwould rapidly and with high fidelity determine the presence and speciesidentification of one or more Candida species in the blood sample; apositive result indicates Candida infection in the patient.

Species identification could rapidly assist in appropriateadministration of antifungals as several Candida species are moreresistant to fluconazole (e.g., Candida glabrata and Candida krusei).

Example 21: Use of the Molecular Beacon Assay to Detect Candida in aWhole Blood Sample

The Candida specific molecular beacon probes described herein can beused to detect the presence of Candida in a whole blood sample from apatient suspected of having a Candida infection. Generally, the protocolinvolves i) obtaining a 1-10 mL sample of whole blood from the patient;ii) lysing the red blood cells (e.g., using an ammonium chlorideiso-osmotic solution, a detergent lysis solution, or a hypotonic lysissolution); iii) centrifuging the lysed sample, e.g., at 3,000 g to12,000 g for 5 minutes to pellet the Candida cells; iv) removing anddiscarding the supernatant; v) washing the pellet with an equal volumeof TE (equivalent to the blood specimen volume) or less; vi)centrifuging the resuspended Candida cells at 3,000 g to 12,000 g for 5minutes to pellet the Candida cells; vii) removing and discarding thesupernatant; viii) optionally repeating steps v)-vii); ix) adding 50-150uL of 1×TE; x) adding 300 mg of 500 to 800 micron beads (e.g., silica orzirconium oxide (yttrium stabilized) beads); xi) vortexing at maximumpower (3000 rpm) for 5-10 minutes to lyse the Candida cells; xii)aliquoting 10 to 50 uL of the cell lysate into a molecular beaconprobe-containing PCR mastermix on, e.g., a 96 well plate (see, e.g.,Example 1); and xiii) running an RT-PCR reaction and detecting thepresence of Candida according to one or more of the methods described inExamples 1-19.

Example 22: Use of the Molecular Beacon Assay to Detect Candida in aUrine Sample

The Candida specific molecular beacon probes described herein can beused to detect the presence of Candida in a urine sample from a patientsuspected of having a Candida infection. Generally, the protocolinvolves i) obtaining a 1-10 mL sample of urine from the patient; ii)centrifuging the sample at 3,000 g to 12,000 g to pellet the Candidacells; iii) removing and discarding the supernatant; iv) washing thepellet with an equal volume of TE or less (equivalent to the urinespecimen volume); v) centrifuging at 3,000 g to 12,000 g for 5 minutesto pellet the Candida cells; vi) removing and discarding thesupernatant; vii) optionally repeating steps iv)-vi); viii) adding50-150 uL of 1×TE; ix) adding 300 mg of 500 to 800 micron beads (e.g.,silica or zirconium oxide (yttrium stabilized) beads); x) vortexing atmaximum power (3000 rpm) for 5-10 minutes to lyse the Candida cells; xi)aliquoting 10 to 50 uL of cell lysate into molecular beaconprobe-containing PCR mastermix on, e.g., a 96 well plate (see, e.g.,Example 1); and xii) running an RT-PCR reaction and detecting thepresence of Candida according to one or more of the methods described inExamples 1-19.

OTHER EMBODIMENTS

While certain novel features of this invention shown and described beloware pointed out in the annexed claims, the invention is not intended tobe limited to the details specified, since a person of ordinary skill inthe relevant art will understand that various omissions, modifications,substitutions and changes in the forms and details of the inventionillustrated and in its operation may be made without departing in anyway from the spirit of the present invention. No feature of theinvention is critical or essential unless it is expressly stated asbeing “critical” or “essential.”

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed in the scope of the present invention.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each independent publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

The invention claimed is:
 1. A nucleic acid probe comprising a sequencehaving at least 95% sequence identity to the sequence of SEQ ID NO: 1conjugated to a detection moiety.
 2. The nucleic acid probe of claim 1,wherein said detection moiety comprises a fluorescent label.
 3. Thenucleic acid probe of claim 2, wherein the fluorescent label is at afirst end of said nucleic acid probe and wherein the nucleic acid probefurther comprises a quencher at an opposite end of said nucleic acidprobe.
 4. The nucleic acid probe of claim 1, wherein the nucleic acidprobe comprises the sequence of SEQ ID NO:
 1. 5. The nucleic acid probeof claim 4, wherein the sequence of the nucleic acid probe consists ofthe sequence of SEQ ID NO:
 1. 6. A composition comprising the nucleicacid probe of claim
 1. 7. The composition of claim 6, further comprisingat least one nucleic acid probe having a sequence with at least 95%sequence identity to a sequence selected from the group consisting ofSEQ ID NOs: 2, 4, and
 5. 8. The composition of claim 7, wherein the atleast one nucleic acid probe has a sequence selected from the groupconsisting of SEQ ID NOs: 2, 4, and
 5. 9. The composition of claim 8,wherein the at least one nucleic acid probe consists of a sequenceselected from the group consisting of SEQ ID NOs: 2, 4, and
 5. 10. Thecomposition of claim 6, wherein the detection moiety comprises afluorescent label.
 11. The composition of claim 10, wherein thefluorescent label is at a first end of the nucleic acid probe andwherein the nucleic acid probe comprises a quencher at an opposite endof the nucleic acid probe.
 12. The composition of claim 6, furthercomprising one or more sample lysis reagents, fungal lysis reagents,fluorescence or nuclear magnetic resonance (NMR)-based reagents fordetection of Candida species, and nucleic acid amplification reagents.13. A kit comprising the composition of claim
 6. 14. The kit of claim13, further comprising one or more sample lysis reagents, fungal lysisreagents, fluorescence or nuclear magnetic resonance (NMR)-basedreagents for detection of Candida species, and nucleic acidamplification reagents.
 15. The composition of claim 6, wherein thenucleic acid probe has the sequence of SEQ ID NO:
 1. 16. The compositionof claim 15, wherein the sequence of the nucleic acid probe consists ofthe sequence of SEQ ID NO:
 1. 17. The composition of claim 15, whereinthe detection moiety comprises a fluorescent label.
 18. The compositionof claim 7, wherein the at least one nucleic acid probe furthercomprises a fluorescent label at a first end of the at least one nucleicacid probe and a quencher at an opposite end of the at least one nucleicacid probe.
 19. The composition of claim 7, further comprising one ormore sample lysis reagents, fungal lysis reagents, fluorescence ornuclear magnetic resonance (NMR)-based reagents for detection of Candidaspecies, and nucleic acid amplification reagents.
 20. The kit of claim13, wherein the nucleic acid probe has the sequence of SEQ ID NO:
 1. 21.The kit of claim 13, further comprising at least one nucleic acid probehaving a sequence with at least 95% sequence identity to a sequenceselected from the group consisting of SEQ ID NOs: 2, 4, and
 5. 22. Thekit of claim 21, wherein the at least one nucleic acid probe has asequence selected from the group consisting of SEQ ID NOs: 2, 4, and 5.23. The kit of claim 22, wherein the at least one nucleic acid probeconsists of a sequence selected from the group consisting of SEQ ID NOs:2, 4, and 5.